US20040210954A1

US20040210954A1 - Integrase mediated avian transgenesis

Info

Publication number: US20040210954A1
Application number: US10/790,455
Authority: US
Inventors: Alex Harvey; Leandro Christmann; Markley Leavitt
Original assignee: Individual
Current assignee: University of Georgia Research Foundation Inc UGARF
Priority date: 2003-03-07
Filing date: 2004-03-01
Publication date: 2004-10-21
Also published as: EP1608219A4; WO2004080162A2; WO2004080162A3; US20050066383A1; EP1608219A2

Abstract

The invention provides methods forintegrating a heterologous polynucleotide into the genome of an avian cell. The methods deliver to an avian cell a polynucleotide and a source of integrase activity that mediates recombination between the polynucleotide and the genomic DNA of the avian cell. The invention provides modified avian or artificial chromosomes as vectors to shuttle transgenes or gene clusters into an avian genome. Another aspect of the invention are avian cells genetically modified with a transgene vector. One cell line for the delivery and integration of a transgene comprises a heterologous attP site and, optionally, a region for expressing the integrase. Methods are also included for the production of a heterologous polypeptide by transgenic avian tissue involve integrating a heterologous polynucleotide into the avian genome. Subsequently, a mature transgenic avian is derived by transferring the transgenic blastodermal cells to an embryo and allowing that embryo to develop fully.

Description

The present application claims priority from U.S. provisional patent application Ser. Nos. 60/453,126, filed Mar. 7, 2003, 60/490,452, filed Jul. 28, 2003, and 60/536,677 filed Jan. 15, 2004 and which are hereby incorporated by reference in their entireties.[0001]

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, and more specifically to the field of avian genome modification. Disclosed herein are compositions, vectors, and methods of use thereof, for the generation of genetically transformed avian cells and transgenic birds.

BACKGROUND

Transgenic technology to convert animals into “bioreactors” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression. Recombinant nucleic acid molecules, for instance, have been engineered and incorporated into transgenic animals so that an expressed heterologous protein may be joined to a protein or peptide that allows secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures, however, may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, such as cows, sheep, or goats.

Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic, heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (e.g., Krimpenfort et al., U.S. Pat. No. 5,175,384).

One system that holds potential is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infindibulum of the oviduct where it is fertilized if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk. The hen oviduct offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species.

One method for creating permanent genomic modification of an eukaryotic cell is to integrate an introduced DNA into an existing chromosome. Only retroviruses have so far provided efficient integration. However, retroviral integration is directed to a number, albeit limited, of insertion sites within the recipient genome so that positional variation in heterologous gene expression can be evident. Unpredictability as to which insertion site is targeted introduces an undesirable lack of control over the procedure. An additional limitation of the use of retroviruses is that the size of the nucleic acid molecule encoding the virus and heterologous sequences is restricted to about 8 kb. Although wild-type adeno-associated virus (AAV) often integrates at a specific region in the human genome, vectors derived from AAV do not integrate site-specifically due to the deletion of the toxic rep gene. Other well-known methods for genomic modification of animal cells include transfection of DNA using calcium phosphate co-precipitation, electroporation, lipofection, microinjection, protoplast fusion and particle bombardment, all of which methods typically produce random integration and at low frequency. Homologous recombination produces site-specific integration, but the frequency of such integration usually is very low.

An alternative method that has been considered for driving the integration of heterologous nucleic acid fragments into a chromosome is the use of a site-specific recombinase (integrase) that can catalyze the insertion or excision of nucleic acid fragments. These enzymes recognize relatively short unique nucleic acid sequences that serve for both recognition and recombination. Examples include Cre (Sternberg & Hamilton, 1981, J. Mol. Biol. 150: 467-486, 1981), Flp (Broach et al., 1982, Cell 29: 227-234, 1982) and R (Matsuzaki et al., 1990, J. Bact. 172: 610-618, 1990).

A novel class of phage integrases that includes the integrase from the phage phiC31 can mediate highly efficient integration of transgenes in mammalian cells both in vitro and in vivo (Thyagarajan et al., Mol. Cell Biol. 21: 3926-3934 (2001)). Constructs and methods of using recombinase to integrate heterologous DNA into a plant, insect or mammalian genome are described by Calos in U.S. patent Ser. No. 6,632,672.

The phiC31 integrase is a member of a subclass of integrases, termed serine recombinases, that include R4 and TP901-1. Unlike the phage lambda integrases, which belong to a tyrosine class of recombinases, the serine integrases do not require cofactors such as integration host factor. The phiC31 integrase normally mediates integration of the phiC31 bacteriophage into the genome of Streptomyces via recombination between the attP recognition sequence of the phage genome and the attB recognition sequence within the bacterial genome. When a plasmid is equipped with a single attB site, phiC31 integrase will detect and mediate crossover between the attB site and a pseudo-attP site within the mammalian genome. Such pseudo-attP integration sites have now been identified in the mouse and human genomes. If the heterologous DNA is in a circular or supercoiled form, the entire plasmid becomes integrated with attL and attR arms flanking the nucleic acid insert. PhiC31 integrase is not able to mediate the integration into genomic DNA of sequences bearing attP sites.

PhiC31 integrase-mediated integration results in the destruction of the recognition or recombination sites themselves so that the integration reaction is irreversible. This will bypass the primary concern inherent with other recombinases, i.e., the reversibility of the integration reaction and excision of the inserted DNA.

It has been estimated that there are 50 to 100 pseudo-attP sites in mammalian genomes (mouse and human) and some sites are apparently preferred for integration over others. The chicken genome, however, is only about one-third the size of mammalian genomes, and it was unknown whether there would be a sufficient number of pseudo attP sites in the chicken genome to allow efficient integrase-mediated integration.

We have found that the phiC31 integrase is active in avian cells, increasing the rate of integration over that of a non-integrase-mediated integration. Furthermore, we have determined that the phiC31 integrase works well at both 37° Celsius and 41° Celsius, showing that it will function in the environment of a developing avian embryo.

A need still exists, however, for methods by which avian chromosomes can be permanently modified in an efficient and site-specific manner and the genetically transformed cells used to generate transgenic birds.

SUMMARY OF THE INVENTION

Integration of a transgene into a defined chromosomal site is useful to improve the predictability of expression of the transgene, which is particularly advantageous when creating transgenic avians. Transgenesis by methods that randomly insert a transgene into an avian genome is often inefficient since the transgene may not be expressed at the desired levels or in desired tissues.

A novel class of phage integrases, and in particular the integrase from phage phiC31, can mediate the efficient integration of transgenes into target cells both in vitro and in vivo. When a plasmid is equipped with a single attB site, phiC31 integrase detects attP homologous sequences, termed pseudo-attP sites, in a target genome and mediates crossover between the attB site and a pseudo attP site.

The present invention provides novel methods and recombinant polynucleotide molecules for transfecting and integrating a heterologous nucleic acid molecule into the genome of an avian cell. The methods of the invention deliver to an avian cell population a first nucleic acid molecule that comprises a region encoding a bacterial recombination site. A source of integrase activity also delivered top the avian cell can be an integrase-encoding nucleic acid sequence and its associated promoter included in the first nucleic acid molecule or as a region of a second nucleic acid molecule that may be co-delivered with the polynucleotide molecule. Alternatively, integrase protein itself can be delivered directly to the target cell.

The recombinant nucleic acid molecules of the present invention may further comprise a heterologous nucleotide sequence operably linked to a promoter so that the heterologous nucleotide sequence, when integrated into the genome DNA of a recipient avian cell, can be expressed to yield a desired polypeptide. The nucleic acid molecule may also include a second transcription initiation site, such as an internal ribosome entry site (IRES), operably linked to a second heterologous polypeptide-encoding region desired to be expressed with the first polypeptide in the same cell.

The heterologous nucleic acid molecule of the present invention may include a cassette for the expression in a recipient avian cell of a desired heterologous polypeptide. Optionally, the nucleic acid molecules may further comprise a marker such as, but not limited to, a puromycin resistance gene, a luciferase gene, EGFP-encoding gene, and the like.

Once delivered to a recipient avian cell, the phiC31 integrase mediates recombination between the att site within the nucleic acid molecule and a bacteriophage attachment site within the genomic DNA of the avian cell. Both att sites are disrupted and the nucleic acid molecule, with partial att sequences at each end, is stably integrated into the genome attP site. The phiC31 integrase, by disrupting the att sites of the incoming nucleic acid and of the recipient site within the avian cell genome, precludes any subsequent reverse recombination event that would excise the integrated nucleic acid and reduce the overall efficiency of stable incorporation of the heterologous nucleic acid.

Following delivery of the nucleic acid molecule and a source of integrase activity into an avian cell population and integrase-mediated recombination, the cells may be returned to an embryo. Late stage blastodermal cells may be returned to a hard shell egg, which is resealed for incubation until hatching. Stage I cells may be directly microinjected with the polynucleotide and source of integrase activity, or isolated, transfected and returned to a stage I embryo which is reimplanted into a hen for further development. Alternatively, the transfected cells may be maintained in in vitro culture.

The present invention further provides modified isolated avian or artificial chromosomes useful as vectors to shuttle transgenes or gene clusters into the avian genome. By delivery to the modified chromosome to an isolated recipient cell, the target cell, and progeny thereof, become trisomic. The additional or trisomic chromosome will not affect the subsequent development of the recipient cell and/or an embryo, nor interfere with the reproductive capacity of an adult bird developed from such cells or embryos. The chromosome will also be stable within chicken cells. The invention provides methods to isolate a population of chromosomes for delivery into chicken embryos or early cells.

The method comprises inserting a lac-operator sequence into an isolated chromosome and, optionally, inserting a desired transgene sequence within the same chromosome. The lac operator region is typically a concatamer of a plurality of lac operators for the binding of multiple lac repressor molecules. A recombinant DNA molecule is constructed that includes an identified region of the target chromosome, a recombination site such as attB or attP, and the lac-operator concatamer. The recombinant molecule is delivered to an avian cell, and homologous recombination will integrate the heterologous polynucleotide and the lac-operator concatamer into the targeted chromosome. A tag-polypeptide, such as the GPF-lac-repressor fusion protein, binds to the lac-operator sequence for identification and isolation of the genetically modified chromosome. The tagged mitotic chromosome can be isolated using, for instance, flow cytometry.

Another aspect of the present invention is an avian cell genetically modified with a transgene vector by the methods of the invention. For example, in one embodiment, the transformed cell can be a chicken early stage blastodermal cell or a genetically transformed cell line, including a sustainable cell line. The transfected cell may comprise a transgene stably integrated into the nuclear genome of the recipient cell, thereby replicating with the cell so that each progeny cell receives a copy of the transfected nucleic acid. A particularly useful cell line for the delivery and integration of a transgene comprises a heterologous attP site that can increase the efficiency of integration of a polynucleotide by phiC31 integrase and, optionally, a region for expressing the integrase.

Another aspect of the present invention is methods of expressing a heterologous polypeptide in an avian cell by stably transfecting a cell by using site-specific integrase-mediation and a recombinant nucleic acid molecule, as described above, and culturing the transfected cell under conditions suitable for expression of the heterologous polypeptide under the control of the avian transcriptional regulatory region.

Yet another aspect of the present invention concerns transgenic birds, such as chickens, comprising a recombinant nucleic acid molecule and which preferably (though optionally) express a heterologous gene in one or more cells in the animal. Embodiments of the methods for the production of a heterologous polypeptide by the avian tissue involve providing a suitable vector and introducing the vector into embryonic blastodermal cells together with an integrase, preferably phiC31 integrase, so that the vector can integrate into the avian genome. A subsequent step involves deriving a mature transgenic avian from the transgenic blastodermal cells by transferring the transgenic blastodermal cells to an embryo and allowing that embryo to develop fully, so that the cells become incorporated into the bird as the embryo is allowed to develop. An alternative is to transfer a transfected nucleus to an enucleated recipient cell which may then develop into a zygote and ultimately an adult bird. The resulting chick is then grown to maturity.

In various embodiments of the transgenic bird of the present invention, the expression of the transgene may be restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, trans-acting factors acting on the transcriptional regulatory region operably linked to the polypeptide-encoding region of interest of the present invention and which control gene expression in the desired pattern. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.

The invention can be used to express, in large yields and at low cost, a wide range of desired proteins including those used as human and animal pharmaceuticals, diagnostics, and livestock feed additives. Proteins such as growth hormones, cytokines, structural proteins and enzymes including human growth hormone, interferon, lysozyme, and β-casein are examples of proteins which are desirably expressed in the oviduct and deposited in eggs according to the invention.

Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates phage integrase-mediated integration. A plasmid vector bearing the transgene includes the attB recognition sequence for the phage integrase. The vector along with integrase-coding mRNA, a vector expressing the integrase, or the integrase protein itself, are delivered into cells or embryos. The integrase recognizes DNA sequences in the avian genome similar to attP sites, termed pseudo-attP, and mediates recombination between the attB and pseudo-attP sites, resulting in the permanent integration of the transgene into the avian genome. [0029]
FIG. 2 illustrates the persistent expression of luciferase from a nucleic acid molecule after phiC31 integrase-mediated integration into chicken cells. [0030]
FIG. 3 illustrates the results of a puromycin resistance assay to measure phiC31 integrase-mediated integration into chicken cells. [0031]
FIG. 4 illustrates phiC31 integrase-mediated integration into quail cells. Puromycin resistance vectors bearing attB sites were cotransfected with phiC31 integrase, or a control vector, into QT6 cells, a quail fibrosarcoma cell line. One day after transfection, puromycin was added. Puromycin resistant colonies were counted 12 days post-transfection. [0032]
FIGS. 5A and 5B illustrate that phiC31 integrase can facilitate multiple integrations per avian cell. A puromycin resistance vector bearing an attB site was cotransfected with an enhanced green fluorescent protein (EGFP) expression vector bearing an attB site, and a phiC31 integrase expression vector. After puromycin selection, many puromycin resistant colonies expressed EGFP in all of their cells. FIGS. 5A and 5B are the same field of view with EGFP illuminated with ultraviolet light (FIG. 5A) and puromycin resistant colonies photographed in visible light (FIG. 5B). In FIG. 5B, there are 4 puromycin resistant colonies, two of which are juxtaposed at the top. One of these colonies expressed EGFP. [0033]
FIG. 6 shows maps of the small vectors used for integrase assays. [0034]
FIG. 7 shows integrase promotes efficient integration of large transgenes in avian cells. [0035]
FIG. 8 shows maps of large vectors used for integrase assays. [0036]
FIG. 9 illustrates the nucleotide sequence of the integrase-expressing plasmid pCMV-31int (SEQ ID NO: 1). [0037]
FIG. 10 illustrates the nucleotide sequence of the plasmid pCMV-luc-attB (SEQ ID NO: 2). [0038]
FIG. 11 illustrates the nucleotide sequence of the plasmid pCMV-luc-attP (SEQ ID NO: 3). [0039]
FIG. 12 illustrates the nucleotide sequence of the plasmid pCMV-pur-attB (SEQ ID NO: 4). [0040]
FIG. 13 illustrates the nucleotide sequence of the plasmid pCMV-pur-attP (SEQ ID NO: 5). [0041]
FIG. 14 illustrates the nucleotide sequence of the plasmid pCMV-EGFP-attB (SEQ ID NO: 6). [0042]
FIG. 15 illustrates the nucleotide sequence of the plasmid p12.0-lys-LSPIPNMM-CMV-pur-attB (SEQ ID NO: 7). [0043]
FIG. 16 illustrates the nucleotide sequence of the plasmid pOMIFN-Ins-CMV-pur-attB (SEQ ID NO: 8). [0044]
FIG. 17 illustrates the nucleotide sequence of the integrase-expressing plasmid pRSV-Int (SEQ ID NO: 9). [0045]
FIG. 18 illustrates the nucleotide sequence of the plasmid pCR-XL-TOPO-CMV-pur-attB (SEQ ID NO: 10). [0046]
FIG. 19 illustrates the nucleotide sequence of the attP containing polynucleotide SEQ ID NO: 11. [0047]
FIG. 20 illustrates in schematic form the integration of a heterologous att recombination site into an isolated chromosome. The attB sequence is linked to selectable maker such as a puromycin expression cassette and is flanked by sequences found in the target site of the chromosome to be modified. The DNA is transfected into cells containing the chromosome and stable transfectants are selected by drug resistance. Site specific integration may be confirmed by several techniques including PCR. [0048]
FIG. 21 illustrates the persistent expression of luciferase from a nucleic acid molecule after phiC31 integrase-mediated integration into chicken cells bearing a wild-type attP sequence.[0049]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This description uses gene nomenclature accepted by the Cucurbit Genetics Cooperative as it appears in the [0050] Cucurbit Genetics Cooperative Report 18:85 (1995), which are incorporated herein by reference in its entirety. Using this gene nomenclature, genes are symbolized by italicized Roman letters. If a mutant gene is recessive to the normal type, then the symbol and name of the mutant gene appear in italicized lower case letters.
The disclosures of publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. [0051]
Definitions [0052]
For convenience, definitions of certain terms employed in the specification, examples, and appended claims are collected here. [0053]
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an antigen” includes a mixture of two or more such agents. [0054]
The term “avian” as used herein refers to any species, subspecies or race of organism of the taxonomic class ava, such as, but not limited to chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of [0055] Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities. It also includes an individual avian organism in all stages of development, including embryonic and fetal stages. The term “avian” also may denote “pertaining to a bird”, such as “an avian (bird) cell.”
The term “nucleic acid” as used herein refers to any natural or synthetic linear and sequential array of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. For ease of discussion, such nucleic acids may be collectively referred to herein as “constructs,” “plasmids,” or “vectors.” The term “nucleic acid” further includes modified or derivatized nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled nucleotides. [0056]
The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid sequence” are used interchangeably herein and include, but are not limited to, coding sequences (polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences); control sequences (e.g., translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and the like); and regulatory sequences (DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression)). No limitation as to length or to synthetic origin are suggested by the terms described above. [0057]
As used herein the terms “peptide,” “polypeptide” and “protein” refer to a polymer of amino acids in a serial array, linked through peptide bonds. A “peptide” typically is a polymer of at least two to about 30 amino acids linked in a serial array by peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptides” contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology (isolated from an appropriate source such as a bird), or synthesized. The term “polypeptides” further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling moieties. [0058]
The terms “percent sequence identity” or “percent sequence similarity” as used herein refer to the degree of sequence identity between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of Karlin & Attschul, [0059] Proc. Natl. Acad. Sci. 87: 2264-2268 (1990), modified as in Karlin & Attschul, Proc. Natl. Acad. Sci. 90: 5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al., 1990, T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al., Nucl. Acids Res. 25: 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons. Examples of preferred algorithms are FASTA and BESTFIT.
The terms “recombinant nucleic acid” and “recombinant DNA” as used herein refer to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences may include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, protein-encoding sequences and the like. The term “recombinant polypeptide” is meant to include a polypeptide produced by recombinant DNA techniques. A recombinant polypeptide may be distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. [0060]
The term “gene” or “genes” as used herein refers to nucleic acid sequences that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Genes that are not naturally part of a particular organism's genome are referred to as “foreign genes,” “heterologous genes” or “exogenous genes” and genes that are naturally a part of a particular organism's genome are referred to as “endogenous genes”. The term “gene product” refers to an RNA or protein that is encoded by the gene. “Endogenous gene products” are RNAs or proteins encoded by endogenous genes. “Heterologous gene products” are RNAs or proteins encoded by “foreign, heterologous or exogenous genes” and are, therefore, not naturally expressed in the cell. [0061]
The term “expressed” or “expression” as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term “expressed” or “expression” as used herein may also refer to the translation from an RNA molecule to give a protein, a polypeptide or a portion thereof. [0062]
The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. [0063]
The term “transcription regulatory sequences” as used herein refers to nucleotide sequences that are associated with a gene nucleic acid sequence and which regulate the transcriptional expression of the gene. Exemplary transcription regulatory sequences include enhancer elements, hormone response elements, steroid response elements, negative regulatory elements, and the like. [0064]
The term “promoter” as used herein refers to the DNA sequence that determines the site of transcription initiation by an RNA polymerase. A “promoter-proximal element” is a regulatory sequence generally within about 200 base pairs of the transcription start site. [0065]
The term “internal ribosome entry sites (IRES)” as used herein refers to a region of a nucleic acid, most typically an RNA molecule, wherein eukaryotic initiation of protein synthesis occurs far downstream of the 5′ end of the RNA molecule. A 43S pre-initiation complex comprising the elf2 protein bound to GTP and Met-tRNA[0066] _i ^Met, the 40S ribosomal subunit, and factors elf3 and 31flA may bind to an “IRES” before locating an AUG start codon. An “IRES” may be used to initiate translation of a second coding region downstream of a first coding region, wherein each coding region is expressed individually, but under the initial control of a single upstream promoter. An “IRES” may be located in a eukaryotic cellular mRNA.
The term “coding region” as used herein refers to a continuous linear arrangement of nucleotides which may be translated into a polypeptide. A full length coding region is translated into a full length protein; that is, a complete protein as would be translated in its natural state absent any post-translational modifications. A full length coding region may also include any leader protein sequence or any other region of the protein that may be excised naturally from the translated protein. [0067]
The terms “vector” or “nucleic acid vector” as used herein refer to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule (RNA or DNA) that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. The term “expression vector” as used herein refers to a nucleic acid vector that comprises a transcription regulatory region operably linked to a site wherein is, or can be, inserted, a nucleotide sequence to be transcribed and, optionally, to be expressed, for instance, but not limited to, a sequence coding at least one polypeptide. [0068]
The term “transfection” as used herein refers to the process of inserting a nucleic acid into a host cell. Many techniques are well known to those skilled in the art to facilitate transfection of a nucleic acid into an eukaryotic cell. These methods include, for instance, treating the cells with high concentrations of salt such as a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules, and by such methods as micro-injection into a pro-nucleus, sperm-mediated and restriction-mediated integration. [0069]
The terms “recombinant cell” and “genetically transformed cell” refer to a cell comprising a combination of nucleic acid segments not found in a single cell with each other in nature. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The recombinant cell may harbor a vector that is extragenomic, i.e.that does not covalently insert into the cellular genome, including a non-nuclear (e.g. mitochondrial) genome(s). A recombinant cell may further harbor a vector or a portion thereof that is intragenomic, i.e. covalently incorporated within the genome of the recombinant cell. [0070]
As used herein, a “transgenic avian” is any avian, as defined above, including the chicken and quail, in which one or more of the cells of the avian contain heterologous nucleic acid introduced by manipulation, such as by transgenic techniques. The nucleic acid may be introduced into a cell, directly or indirectly, by introduction into a precursor of the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. Genetic manipulation also includes classical cross-breeding, or in vitro fertilization. A recombinant DNA molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. [0071]
The terms “chimeric animal” or “mosaic animal” are used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed, in some but not all cells of the animal. The term “tissue-specific chimeric animal” indicates that the recombinant gene is present and/or expressed in some tissues but not others. [0072]
As used herein, the term “transgene” means a nucleic acid sequence that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). [0073]
The term “cytokine” as used herein refers to any secreted polypeptide that affects a function of cells and modulates an interaction between cells in the immune, inflammatory or hematopoietic response. A cytokine includes, but is not limited to, monokines and lymphokines. Examples of cytokines include, but are not limited to, interferon α2b, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-α (TNF-α.) and Tumor Necrosis Factor β (TNF-β.). [0074]
The term “antibody” as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof. Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)[0075] ₂fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
The term “immunoglobulin polypeptide” as used herein refers to a constituent polypeptide of an antibody or a polypeptide derived therefrom. An “immunological polypeptide” may be, but is not limited to, an immunological heavy or light chain and may include a variable region, a diversity region, joining region and a constant region or any combination, variant or truncated form thereof. The term “immunological polypeptides” further includes single-chain antibodies comprised of, but not limited to, an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region and optionally a peptide linker. [0076]
The terms “integrase” and “integrase activity” as used herein refer to a nucleic acid recombinase of the serine recombinase family of proteins. [0077]
The term “source of integrase activity” as used herein refers to a polypeptide or multimeric protein having serine recombinase (integrase) activity in an avian cell. The term may further refer to a polynucleotide encoding the serine recombinase, such as an mRNA, an expression vector, a gene or isolated gene that may be expressed as the recombinase-specific polypeptide or protein. [0078]
The term “recombination site” as used herein refers to a polynucleotide stretch comprising a recombination site normally recognized and used by an integrase. For example, λ phage is a temperate bacteriophage that infects [0079] E. coli. The phage has one attachment site for recombination (attP) and the E. coli bacterial genome has an attachment site for recombination (attB). Both of these sites are recombination sites for λ integrase. Recombination sites recognized by a particular integrase can be derived from a homologous system and associated with heterologous sequences, for example, the attP site can be placed in other systems to act as a substrate for the integrase.
The term “pseudo-recombination site” as used herein refers to a site at which an integrase can facilitate recombination even though the site may not have a sequence identical to the sequence of its wild-type recombination site. For example, a phiC31 integrase and vector carrying a phiC31 wild-type recombination site can be placed into an avian cell. The wild-type recombination sequence aligns itself with a sequence in the avian cell genome and the integrase facilitates a recombination event. When the sequence from the genomic site in the avian cell, where the integration of the vector took place, is examined, the sequence at the genomic site typically has some identity to, but may not be identical with, the wild-type bacterial genome recombination site. The recombination site in the avian cell genome is considered to be a pseudo-recombination site (e.g., a pseudo-attP site) at least because the avian cell is heterologous to the normal phiC31 phage/bacterial cell system. The size of the pseudo-recombination site can be determined through the use of a variety of methods including, but not limited to, (i) sequence alignment comparisons, (ii) secondary structural comparisons, (iii) deletion or point mutation analysis to find the functional limits of the pseudo-recombination site, and (iv) combinations of the foregoing. [0080]
A nucleic acid fragment of interest may be a trait-producing sequence, by which it is meant a sequence conferring a non-native trait upon the cell in which the protein encoded by the trait-producing sequence is expressed. The term “non-native” when used in the context of a trait-producing sequence means that the trait produced is different than one would find in an unmodified organism which can mean that the organism produces high amounts of a natural substance in comparison to an unmodified organism, or produces a non-natural substance. For example, the genome of a bird could be modified to produce proteins not normally produced in birds such as, for instance, human or mouse antibodies, human cytokines, etc. Other useful traits include disease resistance, meat flavor, animal size, and the like. [0081]
A nucleic acid fragment of interest may additionally be a “marker nucleic acid” or expressed as a “marker polypeptide”. Marker genes encode proteins that can be easily detected in transformed cells and are, therefore, useful in the study of those cells. Examples of suitable marker genes include β-galactosidase, green or yellow fluorescent proteins, enhanced green fluorescent protein, chloramphenicol acetyl transferase, luciferase, and the like. Such regions may also include those 5′ noncoding sequences involved with initiation of transcription and translation, such as the enhancer, TATA box, capping sequence, CAAT sequence, and the like [0082]
The term “transformed” as used herein refers to a heritable alteration in a cell resulting from the uptake of a heterologous DNA. [0083]
The term “trisomic” as used herein refers to a cell or animal, such as an avian cell or bird that has a 2n+1 chromosomal complement, where n is the haploid number of chromosomes, for the animal species concerned. [0084]
Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols without undue experimentation. See, for example, Sambrook et al, 1989, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety. [0085]
Abbreviations [0086]
Abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); kb, kilobase; att, bacterial recombination attachment site; IU, infectious units. [0087]
In the standard method of integrase mediated-transgenesis, a serine recombinase integrase mediates recombination between an attB site on a transgene vector and a pseudo attP site on a chromosome. In the method of the invention for integrase-mediated transgenesis, a heterologous wild-type attP site can be integrated into an avian nuclear genome to create a transgenic cell line or bird. A serine recombinase (integrase) and an attB-bearing transgene vector are then introduced into cells harboring the heterologous attP site, or into embryos derived from birds which bear the attP recombination site. The locations of attP and attB may be reversed such that the attB site is inserted into an avian chromosome and the attP sequence resides in an incoming transgene vector. In either case, the att site of the introduced vector would then preferentially recombine with the integrated heterologous att site in the genome of the recipient cell. [0088]
The methods of the invention are based, in part, on the discovery that there exist in avian genomes a number of specific nucleic acid sequences, termed pseudo-recombination sites, the sequences of which may be distinct from wild-type recombination sites but which can be recognized by a site-specific integrase and used to promote the efficient insertion of heterologous genes or polynucleotides into the targeted avian nuclear genome. The inventors have identified pseudo-recombination sites in avian cells capable of recombining with a recombination site, such as an attB site within a recombinant nucleic acid molecule introduced into the target avian cell. The invention is also based on the prior integration of a heterologous att recombination site, typically isolated from a bacteriophage or a modification thereof, into the genome of the target avian cell. [0089]
Integration into a predicted chromosomal site is useful to improve the predictability of expression, which is particularly advantageous when creating transgenic avians. Transgenesis by methods that result in insertion of the transgene into random positions of the avian genome is unpredictable since the transgene may not express at the expected levels or in the predicted tissues. [0090]
The invention as disclosed herein, therefore, provides methods for site-specifically genetically transforming an avian nuclear genome. In general, an avian cell having a first recombination site in the nuclear genome is transformed with a site-specific polynucleotide construct comprising a second recombination sequence and one or more polynucleotides of interest. Into the same cell, integrase activity is introduced that specifically recognizes the first and second recombination sites under conditions such that the polynucleotide sequence of interest is inserted into the nuclear genome via an integrase-mediated recombination event between the first and second recombination sites. [0091]
The integrase activity, or a source thereof, can be introduced into the avian cell prior to, or concurrent with, the introduction of the site-specific construct. The integrase can be delivered to a cell as a polypeptide, or by expressing the integrase from a source polynucleotide such as an mRNA or from an expression vector that encodes the integrase, either of which can be delivered to the target avian cell before, during or after delivery of the polynucleotide of interest. Any integrase that has activity in an avian cell may be useful in the present invention, including HK022 (Kolot et al., [0092] Biotechnol. Bioeng., 84: 56-60 (2003)). Preferably, the integrase is a serine recombinase as described, for example, by Smith & Thorpe, in Mol. Microbiol., 44: 299-307 (2002). More preferably, the integrase is a bacteriophage integrase such as, but not limited to, TP901-1 (Stoll et al., J. Bact., 184: 3657-3663 (2002); Olivares et al., Gene, 278:167-176 (2001). Most preferably, the integrase is from the phage phiC31.
The nucleotide sequence of the junctions between an integrated transgene into the attP (or attB site) would be known. Thus, a PCR assay can be designed by one of skill in the art to detect when the integration event has occurred. The PCR assay for integration into a heterologous wild-type attB or attP site can also be readily incorporated into a quantitative PCR assay using TAQMAN™ or related technology so that the efficiency of-integration can be measured. [0093]
The minimal attB and attP sites able to catalyze recombination mediated by the phiC31 integrase are 34 and 39 bp, respectively. In cell lines that harbor a heterologous integrated attP site, however, integrase has a preference for the inserted attP over any pseudo-attP sites of similar length, because pseudo-attP sites have very low sequence identity (between 10 to 50% identity) compared to the more efficient wild-type attP sequence. It is within the scope of the methods of the invention, however, for the recombination site within the target avian genome to be a pseudo-att site such as a pseudo-attP site or an attP introduced into an avian genome. [0094]
The sites used for recognition and recombination of phage and bacterial DNAs (the native host system) are generally non-identical, although they typically have a common core region of nucleic acids. The bacterial sequence is generally called the attB sequence (bacterial attachment) and the phage sequence is called the attP sequence (phage attachment). Because they are different sequences, recombination will result in a stretch of nucleic acids (called attL or attR for left and right) that is neither an attB sequence or an attP sequence, and likely is functionally unrecognizable as a recombination site to the relevant enzyme, thus removing the possibility that the enzyme will catalyze a second recombination reaction that would reverse the first. [0095]
The integrase may recognize a recombination site where sequence of the 5′ region of the recombination site can differ from the sequence of the 3′ region of the recombination sequence. For example, for the phage phiC31 attP (the phage attachment site), the core region is 5′-TTG-3′ the flanking sequences on either side are represented here as attP5′ and attP3′, the structure of the attP recombination site is, accordingly, attP5′-TTG-attP3′. Correspondingly, for the native bacterial genomic target site (attB) the core region is 5′-TTG-3′, and the flanking sequences on either side are represented here as attB5′ and attB3′, the structure of the attB recombination site is, accordingly, attB5′-TTG-attB3′. After a single-site, phiC31 integrase-mediated recombination event takes place between the phiC31 phage and the bacterial genome, the result is the following recombination product: attB5′-TTG-attP3′{phiC31 vector sequences}attP5′-TTG-attB3′. In the method of invention, the attB site will be within a recombinant nucleic acid molecule that may be delivered to a target avian cell. The corresponding attP (or pseudo-attP) site will be within the avian cell nuclear genome. Consequently, after phiC31 integrase mediated recombination, the recombination product, the nuclear genome with the integrated heterologous polynucleotide will have the sequence attP5′-TTG-attB3′{heterologous polynucleotide}-attB5′-TTG-attP3′. Typically, after recombination the post-recombination recombination sites are no longer able to act as substrate for the phiC31 integrase. This results in stable integration with little or no integrase mediated excision. [0096]
While the preferred recombination site to be included in the recombinant nucleic acid molecules and modified chromosomes of the present invention is the attP site, it is contemplated that any attP-like site may be used if compatible with the attB site. For instance, any pseudo-attP site of the chicken genome may be identified according to the methods of Example 7 below and used as a heterologous att recombination site. Such attP-like sites may have a sequence that is at least 25% identical to SEQ ID NO: 11 as shown in FIG. 19, such as described in Groth et al., [0097] Proc. Natl. Acad. Sci. U.S.A. 97: 5995-6000 (2000) incorporated herein by reference in its entirety. Preferably the selected site will have at least the same degree of efficiency of recombination as the attP site (SEQ ID NO: 11) itself.
In the methods of the present invention, the recipient avian cell population may be an isolated avian cell line such as, for example, DF-1 chicken fibroblasts, chicken DT40 cells or a cell population derived from an early stage embryo such as a chicken stage I or stage X embryo. A particularly useful avian cell population is blastodermal cells isolated from an early stage I embryo or a stage X avian embryo. The methods of the present invention, therefore, include steps for the isolation of blastodermal cells that are then suspended in a cell culture medium or buffer for maintaining the cells in a viable state, and which allows the cell suspension to contact the nucleic acids of the present invention. It is also within the scope of the invention for the nucleic acid construct and the source of integrase activity to be delivered directly to an avian embryo such as a blastodermal layer, or to a tissue layer of an adult bird such as the lining of an oviduct. [0098]
When the recipient avian cell population is isolated from an early stage avian embryo, the embryos must first be isolated. For stage I avian embryos from, for example, a chicken, a fertilized ovum is surgically removed from a bird before the deposition of the outer hard shell has occurred. The nucleic acids for integrating a heterologous nucleic acid into a recipient avian cell genome may then be delivered to isolated embryos by lipofection, microinjection (as described in Example 6 below) or electroporation and the like. After delivery of the nucleic acid, the transfected embryo and its yolk may be deposited into the infundibulum of a recipient hen for the deposition of egg white proteins and a hard shell, and laying of the egg. Stage X avian embryos are obtained from freshly laid fertilized eggs and the blastodermal cells isolated as a suspension of cells in a medium, as described in Example 4 below. Isolated stage X blastodermal cell populations, once transfected, may be injected into recipient stage X embryos and the hard shell eggs resealed according to the methods described in U.S. Pat. No. 6,397,777. [0099]
In the methods of the invention, once a heterologous nucleic acid is delivered to the recipient avian cell, the integrase activity is expressed. The expressed integrase (or injected integrase polypeptide) then mediates recombination between the att site of the heterologous nucleic acid molecule, and the att (or pseudo att) site within the genomic DNA of the recipient avian cell. [0100]
It is within the scope of the present invention for the integrase-encoding sequence and a promoter operably linked thereto to be included in the delivered nucleic acid molecule and that expression of the integrase activity occurs before integration of the heterologous nucleic acid into the avian cell genome. Preferably, the integrase-encoding nucleic acid sequence and associated promoter are in an expression vector that may be co-delivered to the recipient avian cell with the heterologous nucleic acid molecule to be integrated into the recipient genome. [0101]
One suitable integrase expressing expression vector for use in the present invention is pCMV-C31int (SEQ ID NO: 1) as shown in FIG. 9, and described in Groth et al., [0102] Proc. Natl. Acad. Sci. U.S.A. 97: 5995-6000 (2000), incorporated herein by reference in its entirety. In pCMV-C31int, expression of the integrase-encoding sequence is driven by the CMV promoter. However, any promoter may be used that will give expression of the integrase in a recipient avian cell, including operably linked avian-specific gene expression control regions of the avian ovalbumin, lysozyme, ovomucin, ovomucoid gene loci, viral gene promoters, inducible promoters, the RSV promoter and the like.
The recombinant nucleic acid molecules of the present invention for delivery of a heterologous polynucleotide to the genome of a recipient avian cell may comprise a nucleotide sequence encoding the attB attachment site of [0103] Streptomyces ambofaciens as described in Thorpe & Smith, Proc. Natl. Acad. Sci. U.S.A. 95: 5505-5510 (1998). The nucleic acid molecule of the present invention further comprises an expression cassette for the expression in a recipient avian cell of a heterologous nucleic acid encoding a desired heterologous polypeptide. Optionally, the nucleic acid molecules may further comprise a marker such as, but not limited to, a puromycin resistance gene, a luciferase gene, EGFP, and the like.
It is contemplated that the expression cassette for introducing a desired heterologous polypeptide comprises a promoter operably linked to a nucleic acid encoding the desired polypeptide and, optionally, a polyadenylation signal sequence. Exemplary nucleic acids suitable for use in the present invention are more fully described in the examples below. [0104]
In the methods of the present invention, following delivery of the nucleic acid molecule and a source of integrase activity into an avian cell population, the cells are maintained under culture conditions suitable for the expression of the integrase and/or for the integrase to mediate recombination between the recombination site of the nucleic acid and recombination site in the genome of the recipient avian cell. When the recipient avian cell is cultured in vitro, such cells may be incubated at 37° Celsius if the cells are chicken early stage blastodermal cells. They may then be injected into an embryo within a hard shell, which is resealed for incubation until hatching. Alternatively, the transfected cells may be maintained in in vitro culture. [0105]

Site-Specific Nucleic Acid Constructs and Methods of Delivery to an Avian Cell

The present invention provides methods for the site-specific insertion of a heterologous nucleic acid molecule into the nuclear genome of an avian cell by delivering to a target avian cell that has a recombination site in its nuclear genome, a source of integrase activity, a site-specific construct that has another recombination site and a polynucleotide of interest, and allowing the integrase activity to facilitate a recombination event between the two recombination sites, thereby integrating the polynucleotide of interest into the avian nuclear genome. [0106]
(a) Expression vector nucleic acid molecules: A variety of recombinant nucleic acid expression vectors are suitable for use in the practice of the present invention. The site-specific constructs described herein can be constructed utilizing methodologies well known in the art of molecular biology (see, for example, [0107] Ausubel or Maniatis) in view of the teachings of the specification. As described above, the constructs are assembled by inserting into a suitable vector backbone a recombination site such as an attP or an attB site, a polynucleotide of interest operably linked to a gene expression control region of interest and, optionally a sequence encoding a positive selection marker. Polynucleotides of interest can include, but are not limited to, expression cassettes encoding a polypeptide to be expressed in the transformed avian cell or in a transgenic bird derived therefrom. The site-specific constructs are typically circular and may also contain selectable markers, an origin of replication, and other elements.
Any of the vectors of the present invention may also optionally include a sequence encoding a signal peptide that directs secretion of the polypeptide expressed by the vector from the transgenic cells, for instance, from tubular gland cells of the oviduct. This aspect of the invention effectively broadens the spectrum of exogenous proteins that may be deposited in the whites of avian eggs using the methods of the invention. Where an exogenous polypeptide would not otherwise be secreted, the vector bearing the coding sequence can be modified to comprise, for instance, about 60 bp encoding a signal peptide. The DNA sequence encoding the signal peptide is inserted in the vector such that the signal peptide is located at the N-terminus of the polypeptide encoded by the vector. [0108]
The expression vectors of the present invention can comprise an avian transcriptional regulatory region for directing expression of either fusion or non-fusion proteins. With fusion vectors, a number of amino acids are usually added to the desired expressed target gene sequence such as, but not limited to, a polypeptide sequence for thioredoxin. A proteolytic cleavage site may further be introduced at a site between the target recombinant protein and the fusion sequence. Additionally, a region of amino acids such as a polymeric histidine region may be introduced to allow binding of the fusion protein to metallic ions such as nickel bonded to a solid support, for purification of the fusion protein. Once the fusion protein has been purified, the cleavage site allows the target recombinant protein to be separated from the fusion sequence. Enzymes suitable for use in cleaving the proteolytic cleavage site include, but are not limited to, Factor Xa and thrombin. Fusion expression vectors that may be useful in the present invention include pGex (Amrad Corp., Melbourne, Australia), pRIT5 (Pharmacia, Piscataway, N.J.) and pMAL (New England Biolabs, Beverly, Mass.), that fuse glutathione S-transferase, protein A, or maltose E binding protein, respectively, to a desired target recombinant protein. [0109]
Epitope tags are short peptide sequences that are recognized by epitope specific antibodies. A fusion protein comprising a recombinant protein and an epitope tag can be simply and easily purified using an antibody bound to a chromatography resin, for example. The presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself. Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin (HA), the peptide Phe-His-His-Thr-Thr, chitin binding domain, and the like. [0110]
Preferred gene expression control regions for use in avian cells include, but are not limited to, avian specific promoters such as the chicken lysozyme, ovalbumin, or ovomucoid promoters, and the like. Particularly useful are tissue-specific promoters such as avian oviduct promoters that allow for expression and delivery of a heterologous polypeptide to an egg white. [0111]
Viral promoters serve the same function as bacterial or eukaryotic promoters and either provide a specific RNA polymerase in trans (bacteriophage T7) or recruit cellular factors and RNA polymerase (SV40, RSV, CMV). Viral promoters may be preferred as they are generally particularly strong promoters. A preferred promoter for use in avian cells is the RSV promoter. [0112]
Selection markers are valuable elements in expression vectors as they provide a means to select for growth of only those cells that contain a vector. Common selectable marker genes include those for resistance to antibiotics such as ampicillin, puromycin, tetracycline, kanamycin, bleomycin, streptomycin, hygromycin, neomycin, ZEOCIN™, and the like. [0113]
Another element useful in an expression vector is an origin of replication. Replication origins are unique DNA segments that contain multiple short repeated sequences that are recognized by multimeric origin-binding proteins and that play a key role in assembling DNA replication enzymes at the origin site. Suitable origins of replication for use in expression vectors employed herein include [0114] E. coli oriC, colE1 plasmid origin, and the like.
A further useful element in an expression vector is a multiple cloning site or polylinker. Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a vector, for example, downstream of the promoter element. These sites are engineered for convenient cloning of DNA into the vector at a specific position. [0115]
Elements such as the foregoing can be combined to produce expression vectors suitable for use in the methods of the invention. Those of skill in the art will be able to select and combine the elements suitable for use in their particular system in view of the teachings of the present specification. [0116]
(b) Genetically modified avian and artificial chromosomes: The present invention further provides modified chromosomes, either isolated avian or artificial chromosomes, are useful vectors to shuttle transgenes or gene clusters into the avian genome. By delivering the modified or artificial chromosome to an isolated recipient cell, the target cell, and progeny thereof, become trisomic. Preferably, an additional or triosomic chromosome will not affect the subsequent development of the recipient cell and/or an embryo, nor interfere with the reproductive capacity of an adult bird developed from such cells or embryos. The chromosome also should be stable within chicken cells. An effective method is also required to isolate a population of chromosomes for delivery into chicken embryos or early cells. [0117]
A number of artificial chromosomes are useful in the methods of the invention, including, for instance, a human chromosome modified to work as an artificial chromosome in a heterologous species as described, for example, for mice (Tomizuka et al., [0118] Proc. Natl. Acad. Sci. U.S.A. 97: 722-727 (2000); for cattle (Kuroiwa et al., Nat. Biotechnol. 20: 889-894 (2002); a mammalian artificial chromosome used in mice (Co et al., Chromosome Res. 8: 183-191 (2000), or in viable triploid chickens (Thorne et al., Cytogenet. Cell Genet. 57: 206-210 (1991) and Thorne, et al., J. Hered. 88: 495-498 (1997). Chickens that are trisomic for microchromosome 16 have been described (Miller et al., Proc. Natl. Acad. Sci. U.S.A. 93: 3958-3962 (1996); Muscarella et al., J. Cell Biol. 101: 1749-1756 (1985). In these cases, triploidy and trisomy occurred naturally, and illustrate that an extra copy of one or more of the chicken chromosomes is compatible with normal development and reproductive capacity.
A useful chromosome isolation protocol can comprise the steps of inserting a lac-operator sequence (Robinett et al. [0119] J. Cell Biol. 135: 1685-1700 (1996) into an isolated chromosome and, optionally, inserting a desired transgene sequence within the same chromosome. Preferably, the lac operator region is a concatamer of a plurality of lac operators for the binding of multiple lac repressor molecules. Insertion can be accomplished, for instance, by identifying a region of known nucleotide sequence associated with a particular avian chromosome. A recombinant DNA molecule may be constructed that comprises the identified region, a recombination site such as attB or attP and a lac-operator concatamer. The recombinant molecule is delivered to an isolated avian cell, preferably, but not limited to, chicken DT40 cells that have elevated homologous recombination activity compared to other avian cell lines, whereupon homologous recombination will integrate the heterologous recombination site and the lac-operator concatamer into the targeted chromosome as shown in the schema illustrated in FIG. 20. A tag-polypeptide comprising a label domain and a lac repressor domain is also delivered to the cell, preferably by expression from a suitable expression vector. The nucleotide sequence coding for a GFP-lac-repressor fusion protein (Robinett et al., J. Cell Biol. 135: 1685-1700 (1996)) may be inserted into the same chromosome as the lac-operator insert. The lac repressor sequence, however, can also be within a different chromosome. An inducible promoter may also be used to allow the expression of the GFP-lac-repressor only after chromosome is to be isolated.
Induced expression of the GPF-lac-repressor fusion protein will result in specific binding of the tag fusion polypeptide to the lac-operator sequence for identification and isolation of the genetically modified chromosome. The tagged mitotic chromosome can be isolated using, for instance, flow cytometry as described in de Jong et al. [0120] Cytometry 35: 129-133 (1999) and Griffin et al. Cytogenet. Cell Genet. 87: 278-281 (1999).
A tagged chromosome can also be isolated using microcell technology requiring treatment of cells with the mitotic inhibitor colcemid to induce the formation of micronuclei containing intact isolated chromosomes within the cell. Final separation of the micronuclei is then accomplished by centrifugation in cytochalasin as described by Killary & Fournier in Methods Enzymol. 254: 133-152 (1995). Further purification of microcells containing only the desired tagged chromosome could be done by flow cytometry. It is contemplated, however, that alternative methods to isolate the mitotic chromosomes or microcells, including mechanical isolation or the use of laser scissors and tweezers, and the like. [0121]

Delivery of a Site-Specific Nucleic Acid to a Recipient Avian Cell or Embryo

(a) Delivery of Polynucleotide Constructs. [0122]
Most non-viral methods of gene transfer rely on normal mechanisms used by eukaryotic cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject transcriptional regulatory region and operably linked polypeptide-encoding nucleic acid by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. Modified chromosomes as described above may be delivered to isolated avian embryonic ells for subsequent introduction to an embryo. [0123]
In a representative embodiment, a nucleic acid molecule can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., 1992, [0124] NO Shinkei Geka 20: 547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075, all of which are incorporated herein by reference in their entireties).
In similar fashion, the gene delivery system can comprise an antibody or cell surface ligand that is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180, all of which are incorporated herein by reference in their entireties). It will also be appreciated that effective delivery of the subject nucleic acid constructs via receptor-mediated endocytosis can be improved using agents which enhance escape of genes from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., 1993, [0125] Science 260-926; Wagner et al., 1992, Proc. Natl. Acad. Sci. 89:7934-7938; and Christiano et al., 1993, Proc. Natl. Acad. Sci. 90:2122-2126, all of which are incorporated herein by reference in their entireties). It is further contemplated that a recombinant nucleic acid molecule of the present invention may be delivered to a target host cell by other non-viral methods including by gene gun, microinjection, sperm-mediated transfer, or the like.
In yet another embodiment of the invention, an expression vector that comprises a heterologous attB recombination site and a region encoding a polypeptide deposited into an egg white are delivered to oviduct cells by in vivo electroporation. In this method, the luminal surface of an avian oviduct is surgically exposed. A buffered solution of the expression vector and a source of integrase activity such as a second expression vector expressing integrase (for example pCMV-int) is deposited on the luminal surface. Electroporation electrodes are then positioned on either side of the oviduct wall, the luminal electrode contacting the expression vector solution. After electroporation, the surgical incisions are closed. The electroporation will deliver the expression vectors to some, if not all, treated recipient oviduct cells to create a tissue-specific chimeric animal. Expression of the integrase allows for the integration of the heterologous polynucleotide into the nuclear genomes of recipient oviduct cells. While this method may be used with any bird, a preferred recipient is a chicken due to the size of the oviduct. More preferred is a transgenic bird that has a transgenic attP recombinant site in the nuclear genomes of recipient oviduct cells, thus increasing the efficiency of integration of the expression vector. [0126]
The attB/P integrase system is preferred in the in vivo electroporation method to allow the formation of stable genetically transformed oviduct cells that otherwise progressively lose the heterologous expression vector. [0127]
The stably modified oviduct cells will express the heterologous polynucleotide and deposit the resulting polypeptide into the egg white of a laid egg. For this purpose, the expression vector will further comprise an oviduct-specific promoter such as ovalbumin or ovomucoid operably linked to the desired heterologous polynucleotide. [0128]
(b) Delivery of Chromosomes to Avian Cells. [0129]
Another aspect of the invention is the generation of a trisomic avian cell comprising a genetically modified extra chromosome. The extra chromosome may be an artificial chromosome or an isolated avian chromosome that has been genetically modified. Introduction of the extra chromosome to an avian cell will generate a trisomic cell with 2n+1 chromosomes, where n is the haploid number of chromosomes of a normal avian cell. [0130]
Delivery of an isolated chromosome into an isolated avian cell or embryo can be accomplished in several ways. Isolated mitotic chromosomes or a micronucleus containing an interphase chromosome can be injected into early stage I embryos by cytoplasmic injection. The injected zygote would then be surgically transferred to a recipient hen for the production and laying of a hard shell egg. This hard shell egg would then be incubated until hatching of a chick. [0131]
Isolated microcells can be fused to primordial germ cells (PGCs) isolated from the blood stream of late stage 15 embryos as described by Killary & Fournier in [0132] Methods Enzymol. 254: 133-152 (1995). The PGC/microcell hybrids can then be transplanted into the blood stream of a recipient embryo to produce germline chimeric chickens. (See Naito et al., Mol. Reprod. Dev. 39: 153-161 (1994)). The manipulated eggs would then incubated until hatching of the bird.
Blastodermal cells isolated from stage X embryos can be transfected with isolated mitotic chromosomes. Following in vitro transfection, the cells are transplanted back into stage X embryos as described, for example, in Etches et al., [0133] Poult. Sci., 72: 882-829 (1993), and the manipulated eggs are incubated to hatching.
Stage X blastodermal cells can also be fused with isolated microcells and then transplanted back into to stage X embryos or fused to somatic cells to be used as nuclear donors for nuclear transfer as described by Kuroiwa et al., [0134] Nat. Biotechnol. 20: 889-894 (2002).
Chromosomal vectors, as described above, may be delivered to a recipient avian cell by, for example, microinjection, liposomal delivery or microcell fusion. [0135]

Delivering a Source of Integrase Activity to an Avian Cell

In the methods of the invention, a site-specific integrase is introduced into an avian cell whose genome is to be modified. Methods of introducing functional proteins into cells are well known in the art. Introduction of purified integrase protein can ensure a transient presence of the protein and its activity. Thus, the lack of permanence associated with most expression vectors is not expected to be detrimental. [0136]
The integrase used in the practice of the present invention can be introduced into a target cell before, concurrently with, or after the introduction of a site-specific vector. The integrase can be directly introduced into a cell as a protein, for example, by using liposomes, coated particles, or microinjection, or into the blastodermal layer of an early stage avian embryo by microinjection. A source of the integrase can also be delivered to an avian cell by introducing to the cell an mRNA encoding the integrase and which can be expressed in the recipient cell as an integrase polypeptide. Alternately, a DNA molecule encoding the integrase can be introduced into the cell using a suitable expression vector. [0137]
The present invention provides novel nucleic acid vectors and methods of use that allow the phiC31 integrase to efficiently integrate a heterologous nucleic acid into an avian genome. A novel finding is that the phiC31 integrase is remarkably efficient in avian cells and increases the rate of integration of heterologous nucleic acid at least 30-fold over that of random integration. Furthermore, the phiC31 integrase works equally well at 37° C. and 41° C., indicating that it will function in the environment of the developing avian embryo, as shown in Example 1. [0138]
The site-specific vector components described above are useful in the construction of expression cassettes containing sequences encoding an integrase. One integrase-expressing vector useful in the methods of the invention is pCMV-C31int (SEQ ID NO: 1 as shown in FIG. 9) where the phiC31 integrase is encoded by a region under the expression control of the strong CMV promoter. Another preferred promoter generally useful in avian cells is the RSV promoter as used in SEQ ID NO: 9 shown in FIG. 17. Expression of the integrase is typically desired to be transient. Accordingly, vectors providing transient expression of the integrase are preferred. However, expression of the integrase can be regulated in other ways, for example, by placing the expression of the integrase under the control of a regulatable promoter (i.e., a promoter whose expression can be selectively induced or repressed). [0139]
Transgenic Avian Cells. [0140]
Another aspect of the present invention is an avian cell genetically modified with a transgene vector according to the present invention and described above. For example, in one embodiment, the transformed cell can be a chicken early stage blastodermal cell or a genetically transformed cell line, including a sustainable cell line. The transfected cell according to the present invention may comprise a transgene stably integrated into the nuclear genome of the recipient cell, thereby replicating with the cell so that each progeny cell receives a copy of the transfected nucleic acid. A particularly useful cell line for the delivery and integration of a transgene comprises a heterologous attP site that can increase the efficiency of integration of a polynucleotide by phiC31 integrase and, optionally, a region for expressing the integrase. [0141]
A retroviral vector can be used to deliver the att site into the avian genome since an attP or attB site is less than 300 bp. For example, the attP site can be inserted into the NLB retroviral vector, which is based on the avian leukosis virus genome. A lentiviral vector is a particularly suitable vector because lentiviral vectors can transduce non-dividing cells, so that a higher percentage of cells will have an integrated attP site. [0142]
The lacZ region of NLB is replaced by the attP sequence. A producer cell line would be created by transformation of, for example, the Isolde cell line capable of producing a packaged recombinant NLB-attP virus pseudo-typed with the envA envelope protein. Supernatant from the Isolde NLB-attP line is concentrated by centrifugation to produce high titer preparations of the retroviral vector that can then be used to deliver the attP site to the genome of an avian cell, as described in Example 9 below. [0143]
An attP-containing line of transgenic birds are a source of attP transgenic embryos and embryonic cells. Fertile zygotes and oocytes bearing a heterologous attP site in either the maternal, paternal, or both, genomes can be used for transgenic insertion of a desired heterologous polynucleotide. A transgene vector bearing an attB site, for example, would be injected into the cytoplasm along with either an integrase expression plasmid, mRNA encoding the integrase or the purified integrase protein. The oocyte or zygote is then cultured to hatch by ex ovo methods or reintroduced into a recipient hen such that the hen lays a bard shell egg the next day containing the injected egg. [0144]
In another example, fertile stage VII-XII embryos hemizygous or homozygous for the heterologous attP sequence, are used as a source of blastodermal cells. The cells are harvested and then transfected with a transgene vector bearing an attB site along with a source of integrase. The transfected cells are then injected into the subgerminal cavity of windowed fertile eggs. The chicks that hatch will bear the transgene integrated into the attP site in a percentage of their somatic and germ cells. To obtain fully transgenic birds, chicks are raised to sexual maturity and those that are positive for the transgene in their semen are bred to non-transgenic mates. [0145]
In various embodiments, the genetically engineered cells of the invention may contain an integrase specifically recognizing recombination sites and which is introduced into genetically engineered cells containing a nucleic acid construct of the invention under conditions such that the nucleic acid sequence(s) of interest will be inserted into the nuclear genome. Methods for introducing such an integrase into a cell are described above. [0146]
In some embodiments, the site-specific integrase is introduced into the cell as a polypeptide. In alternative embodiments, the site-specific integrase is introduced into the transgenic cell as a polynucleotide encoding the integrase, such as an expression cassette optionally carried on a transient expression vector, and comprising a polynucleotide encoding the recombinase. [0147]
In one embodiment, the invention is directed to methods of using a vector for site-specific integration of a heterologous nucleotide sequence into the genome of an avian cell, the vector comprising a circular backbone vector, a polynucleotide of interest operably linked to a promoter, and a first recombination site, wherein the genome of the cell comprises a second recombination site and recombination between the first and second recombination sites is facilitated by phiC31 integrase. In certain embodiments, the integrase facilitates recombination between a bacterial genomic recombination site (attB) and a phage genomic recombination site (attP). [0148]
In another embodiment, the invention is directed to an avian cell having a transformed genome comprising an integrated heterologous polynucleotide of interest whose integration, mediated by phiC31 integrase, was into a recombination site native to the avian cell genome and the integration created a recombination-product site comprising the polynucleotide sequence. In yet another embodiment, integration of the polynucleotide was into a recombination site not native to the avian cell genome, but instead into a heterologous recombination site engineered into the avian cell genome. [0149]
In further embodiments, the invention is directed to transgenic birds comprising a modified cell and progeny thereof as described above, as well as methods of producing the same. [0150]
Cells genetically modified to carry a heterologous attB or attP site by the methods of the present invention can be maintained under conditions that, for example, keep them alive but do not promote growth, promote growth of the cells, and/or cause the cells to differentiate or dedifferentiate. Cell culture conditions may be permissive for the action of the integrase in the cells, although regulation of the activity of the integrase may also be modulated by culture conditions (e.g., raising or lowering the temperature at which the cells are cultured). [0151]
One aspect of the invention is a method for generating a genetically modified avian cell, and progeny thereof, using a tagged chromosome, the method comprising the steps of providing an isolated modified chromosome comprising a lac operator region and a first recombination site, delivering the modified chromosome to a avian cell, thereby generating a trisomic avian cell, delivering to the avian cell a source of a tagged polypeptide comprising a fluorescent domain and a lac repressor domain, delivering a source of integrase activity to the avian cell, delivering a polynucleotide comprising a second recombination site and a region encoding a polypeptide to the avian cell, maintaining the avian cell under conditions suitable for the integrase to mediate recombination between the first and second recombination sites, thereby integrating the polynucleotide into the modified chromosome and generating a genetically modified avian cell, expressing the tag polypeptide by the avian cell, allowing the tag polypeptide to bind to the modified chromosome so as to label the modified chromosome, and isolating the modified chromosome by selecting modified chromosomes having a tag polypeptide bound thereto. [0152]
In one embodiment of the invention, the second avian cell is selected from the group consisting of a stage VII-XII blastodermal cell, a stage I embryo, a stage X embryo; an isolated primordial germ cell, an isolated non-embryonic cell, and an oviduct cell. [0153]
In various embodiments, the isolated modified chromosome is an avian chromosome or an artificial chromosome. [0154]
In other embodiments of the invention, the step of providing an isolated modified chromosome comprising a lac operator region and a first recombination site comprises the steps of generating a trisomic avian cell by delivering to an isolated avian cell an isolated chromosome and a polynucleotide comprising a lac operator and a second recombination site, maintaining the trisomic cell under conditions whereby the heterologous polynucleotide is integrated into the chromosome by homologous recombination, delivering to the avian cell a source of a tag polypeptide to label the chromosome, and isolating the labeled chromosome. [0155]
In one embodiment of the invention, the lac operator region is a concatamer of lac operators. In other embodiments of the invention, the tag polypeptide is expressed from an expression vector. [0156]
In one embodiment of the invention, the tag polypeptide is microinjected into the cell. In various embodiments of the invention, the method of delivery of a chromosome to an avian cell is selected from the group consisting of liposome delivery, microinjection, microcell, electroporation and gene gun delivery, or a combination thereof. [0157]
In embodiments of the invention, the fluorescent domain of the tag polypeptide is GFP. [0158]
In another embodiment of the invention, the method further comprises the step of delivering the second avian cell to an avian embryo. The embryo may be maintained under conditions suitable for hatching as a chick. [0159]
In one embodiment of the invention, the second avian cell is maintained under conditions suitable for the proliferation of the cell, and progeny thereof. [0160]
In various embodiments of the invention, the source of integrase activity is delivered to a first avian cell as a polypeptide or expressed from a polynucleotide, said polynucleotide being selected from an mRNA and an expression vector. [0161]
In one embodiment of the invention, the tag polypeptide activity is delivered to the avian cell as a polypeptide or expressed from a polynucleotide operably linked to a promoter. In another embodiment of the invention, the promoter is an inducible promoter. In yet another embodiment of the invention, the integrase is phiC31 integrase and in various embodiments of the invention, the first and second recombination sites are selected from an attB and an attP site, but wherein the first and second sites are not identical. [0162]

Expression of Heterologous Proteins by Site-Specific Genetic Transformation of Avian Cells

Another aspect of the present invention is a method of expressing a heterologous polypeptide in an avian cell by stably transfecting a cell by using site-specific integrase-mediation and a recombinant nucleic acid molecule, as described above, and culturing the transfected cell under conditions suitable for expression of the heterologous polypeptide under the control of the avian transcriptional regulatory region. [0163]
The protein of the present invention may be produced in purified form by any known conventional techniques. For example, chicken cells, an egg or an egg white may be homogenized and centrifuged. The supernatant may then be subjected to sequential ammonium sulfate precipitation and heat treatment. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC or other methods well known in the art of protein purification. [0164]
The methods of the invention are useful for expressing nucleic acid sequences that are optimized for expression in avian cells and which encode desired polypeptides or derivatives and fragments thereof. Derivatives include, for instance, polypeptides with conservative amino acid replacements, that is, those within a family of amino acids that are related in their side chains (commonly known as acidic, basic, nonpolar, and uncharged polar amino acids). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids and other groupings are known in the art (see, for example, “Biochemistry”, 2nd ed, L. Stryer, ed., W.H. Freeman & Co., 1981). Peptides in which more than one replacement has taken place can readily be tested for activity in the same manner as derivatives with a single replacement, using conventional polypeptide activity assays (e.g. for enzymatic or ligand binding activities). [0165]
Regarding codon optimization, if the recombinant nucleic acid molecules are transfected into a recipient chicken cell, the sequence of the nucleic acid insert to be expressed can be optimized for chicken codon usage. This may be determined from the codon usage of at least one, and preferably more than one, protein expressed in a chicken cell according to well known principles. For example, in the chicken the codon usage could be determined from the nucleic acid sequences encoding the proteins such as lysozyme, ovalbumin, ovomucin and ovotransferrin of chicken. Optimization of the sequence for codon usage can elevate the level of translation in avian eggs. [0166]
The present invention provides methods for the production of a protein by an avian cell comprising the steps of maintaining an avian cell, transfecting with a first expression vector and, optionally, a second expression vector, under conditions suitable for proliferation and/or gene expression and such that an integrase will mediate site specific recombination at att sites. The expression vectors may each have a transcription unit comprising a nucleotide sequence encoding a heterologous polypeptide, wherein one polypeptide is an integrase, a transcription promoter, and a transcriptional terminator. The cells may then be maintained under conditions for the expression and production of the desired heterologous polypeptide(s). [0167]
The present invention further relates to methods for gene expression by avian cells from nucleic acid vectors, and transgenes derived therefrom, that include more than one polypeptide-encoding region wherein, for example, a first polypeptide-encoding region can be operatively linked to an avian promoter and a second polypeptide-encoding region is operatively linked to an Internal Ribosome Entry Sequence (IRES). It is contemplated that the first polypeptide-encoding region, the IRES and the second polypeptide-encoding region of a recombinant DNA of the present invention may be arranged linearly, with the IRES operably positioned immediately 5′ of the second polypeptide-encoding region. This nucleic acid construct, when inserted into the genome of an avian cell or a bird and expressed therein, will generate individual polypeptides that may be post-translationally modified and combined in the white of a hard shell bird egg. Alternatively, the expressed polypeptides may be isolated from an avian egg and combined in vitro. [0168]
The invention, therefore, includes methods for producing multimeric proteins including immunoglobulins, such as antibodies, and antigen binding fragments thereof. Thus, in one embodiment of the present invention, the multimeric protein is an immunoglobulin, wherein the first and second heterologous polypeptides are immunoglobulin heavy and light chains respectively. Illustrative examples of this and other aspects of the present invention for the production of heterologous multimeric polypeptides in avian cells are fully disclosed in U.S. patent application Ser. No. 09/877,374, filed Jun. 8, 2001, by Rapp, published as US-2002-0108132-A1 on Aug. 8, 2002, and U.S. patent application Ser. No. 10/251,364, filed Sep. 18, 2002, by Rapp, both of which are incorporated herein by reference in their entirety. [0169]
Accordingly, the invention further provides immunoglobulin and other multimeric proteins that have been produced by transgenic avians of the invention. [0170]
In various embodiments, an immunoglobulin polypeptide encoded by the transcriptional unit of at least one expression vector may be an immunoglobulin heavy chain polypeptide comprising a variable region or a variant thereof, and may further comprise a D region, a J region, a C region, or a combination thereof. An immunoglobulin polypeptide encoded by an expression vector may also be an immunoglobulin light chain polypeptide comprising a variable region or a variant thereof, and may further comprise a J region and a C region. The present invention also contemplates multiple immunoglobulin regions that are derived from the same animal species, or a mixture of species including, but not only, human, mouse, rat, rabbit and chicken. In preferred embodiments, the antibodies are human or humanized. [0171]
In other embodiments, the immunoglobulin polypeptide encoded by at least one expression vector comprises an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region, and a linker peptide thereby forming a single-chain antibody capable of selectively binding an antigen. [0172]
Examples of therapeutic antibodies that can be used in methods of the invention include but are not limited to HERCEPTIN™ (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO™ (abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptor on the platelets for the prevention of clot formation; ZENAPAX™ (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-1 52 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CATIBASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celitech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-a4p7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan); and CAT-152 is a human anti-TGF-β[0173] ₂antibody (Cambridge Ab Tech).

Production of Heterologous Protein by Transgenic Avians

One aspect of the present invention, therefore, concerns transgenic birds, such as chickens, comprising a recombinant nucleic acid molecule and which preferably (though optionally) express a heterologous gene in one or more cells in the animal. Suitable methods for the generation of transgenic avians having heterologous DNA incorporated therein are described, for example, in WO 99/19472 to Ivarie et al.; WO 00/11151 to Ivarie et al.; and WO 00/56932 to Harvey et al., all of which are incorporated herein by reference in their entirety. [0174]
Embodiments of the methods for the production of a heterologous polypeptide by the avian tissue such as the oviduct and the production of eggs which contain heterologous protein involve providing a suitable vector and introducing the vector into embryonic blastodermal cells together with an integrase, preferably phiC31 integrase, so that the vector can integrate into the avian genome. A subsequent step involves deriving a mature transgenic avian from the transgenic blastodermal cells produced in the previous steps. Deriving a mature transgenic avian from the blastodermal cells optionally involves transferring the transgenic blastodermal cells to an embryo and allowing that embryo to develop fully, so that the cells become incorporated into the bird as the embryo is allowed to develop. Another alternative is to transfer a transfected nucleus to an enucleated recipient cell which may then develop into a zygote and ultimately an adult bird. The resulting chick is then grown to maturity. [0175]
In an alternative embodiment, the cells of a blastodermal embryo are transfected or transduced with the vector and integrase directly within the embryo. It is contemplated, for example, that the recombinant nucleic acid molecules of the present invention may be introduced into a blastodermal embryo by direct microinjection of the DNA into a stage X or earlier embryo that has been removed from the oviduct. The egg is then returned to the bird for egg white deposition, shell development and laying. The resulting embryo is allowed to develop and hatch, and the chick allowed to mature. [0176]
In one embodiment, a transgenic bird of the present invention is produced by introducing into embryonic cells such as, for instance, isolated avian blastodermal cells, a nucleic acid construct comprising an attB recombination site capable of recombining with a pseudo-attP recombination site found within the nuclear genome of the organism from which the cell was derived, and a nucleic acid fragment of interest, in a manner such that the nucleic acid fragment of interest is stably integrated into the nuclear genome of germ line cells of a mature bird and is inherited in normal Mendelian fashion. It is also within the scope of the invention that the targeted cells for receiving the transgene have been engineered to have a heterologous attP recombination site integrated into the nuclear genome of the cells, thereby increasing the efficiency of recognition and recombination with a heterologous attB site. [0177]
In either case, the transgenic bird produced from the transgenic blastodermal cells is known as a “founder” Some founders can be chimeric or mosaic birds if, for example, microinjection does not deliver nucleic acid molecules to all of the blastodermal cells of an embryo. Some founders will carry the transgene in the tubular gland cells in the magnum of their oviducts and will express the heterologous protein encoded by the transgene in their oviducts. If the heterologous protein contains the appropriate signal sequences, it will be secreted into the lumen of the oviduct and onto the yolk of an egg. [0178]
Some founders are germ-line founders. A germ-line founder is a founder that carries the transgene in genetic material of its germ-line tissue, and may also carry the transgene in oviduct magnum tubular gland cells that express the heterologous protein. Therefore, in accordance with the invention, the transgenic bird will have tubular gland cells expressing the heterologous protein and the offspring of the transgenic bird will also have oviduct magnum tubular gland cells that express the selected heterologous protein. (Alternatively, the offspring express a phenotype determined by expression of the exogenous gene in a specific tissue of the avian.) The invention can be used to express, in large yields and at low cost, a wide range of desired proteins including those used as human and animal pharmaceuticals, diagnostics, and livestock feed additives. Proteins such as growth hormones, cytokines, structural proteins and enzymes including human growth hormone, interferon, lysozyme, and β-casein are examples of proteins which are desirably expressed in the oviduct and deposited in eggs according to the invention. Other possible proteins to be produced include, but are not limited to, albumin, α-1 antitrypsin, antithrombin III, collagen, factors VIII, IX, X (and the like), fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), feed additive enzymes, somatotropin, and chymotrypsin. Immunoglobulins (shown, for example in Example 10 below) and genetically engineered antibodies, including immunotoxins which bind to surface antigens on human tumor cells and destroy them, can also be expressed for use as pharmaceuticals or diagnostics. [0179]
In various embodiments of the transgenic bird of the present invention, the expression of the transgene may be restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, trans-acting factors acting on the transcriptional regulatory region operably linked to the polypeptide-encoding region of interest of the present invention and which control gene expression in the desired pattern. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences. [0180]
The stably modified oviduct cells will express the heterologous polynucleotide and deposit the resulting polypeptide into the egg white of a laid egg. For this purpose, the expression vector will further comprise an oviduct-specific promoter such as ovalbumin or ovomucoid operably linked to the desired heterologous polynucleotide. [0181]
Another aspect of the present invention provides a method for the production in an avian of an heterologous protein capable of forming an antibody suitable for selectively binding an antigen. This method comprises a step of producing a transgenic avian incorporating at least one transgene, the transgene encoding at least one heterologous polypeptide selected from an immunoglobulin heavy chain variable region, an immunoglobulin heavy chain comprising a variable region and a constant region, an immunoglobulin light chain variable region, an immunoglobulin light chain comprising a variable region and a constant region, and a single-chain antibody comprising two peptide-linked immunoglobulin variable regions. [0182]
In one embodiment of this method, the isolated heterologous protein is an antibody capable of selectively binding to an antigen and which may be generated by combining at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region, preferably cross-linked by at least one disulfide bridge. The combination of the two variable regions generates a binding site that binds an antigen using methods for antibody reconstitution that are well known in the art. [0183]
The present invention also encompasses immunoglobulin heavy and light chains, or variants or derivatives thereof, to be expressed in separate transgenic avians, and thereafter isolated from separate media including serum or eggs, each isolate comprising one or more distinct species of immunoglobulin polypeptide. The method may further comprise the step of combining a plurality of isolated heterologous immunoglobulin polypeptides, thereby producing an antibody capable of selectively binding to an antigen. In this embodiment, for instance, two or more individual transgenic avians may be generated wherein one transgenic produces serum or eggs having an immunoglobulin heavy chain variable region, or a polypeptide comprising such, expressed therein. A second transgenic animal, having a second transgene, produces serum or eggs having an immunoglobulin light chain variable region, or a polypeptide comprising such, expressed therein. The polypeptides from two or more transgenic animals may be isolated from their respective sera and eggs and combined in vitro to generate a binding site capable of binding an antigen. [0184]
The present invention is further illustrated by the following examples, which are provided by way of illustration and should not be construed as limiting. The contents of all references, published patents and patents cited throughout the present application are hereby incorporated by reference in their entireties. [0185]
It will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations as come within the scope of the appended claims and their equivalents. [0186]

EXAMPLE 1

Phage phiC31 Integrase Functions in Avian Cells

(a) A luciferase vector bearing either an attB (SEQ ID NO: 2 shown in FIG. 10) or attP (SEQ ID NO: 3 shown in FIG. 11) site was co-transfected with an integrase expression vector CMV-C31int (SEQ ID NO: 1) into DF-1 cells, a chicken fibroblast cell line. The cells were passaged several times and the luciferase levels were assayed at each passage. [0187]
Cells were passaged every 3-4 days and one third of the cells were harvested and assayed for luciferase. The expression of luciferase was plotted as a percentage of the expression measured 4 days after transfection. A luciferase expression vector bearing an attP site as a control was also included. [0188]
As can be seen in FIG. 2, in the absence of integrase, luciferase expression from a vector bearing attP or attB decreased to very low levels after several days. However, luciferase levels were persistent when the luciferase vector bearing attB was co-transfected with the integrase expression vector, indicating that the luciferase vector had stably integrated into the avian genome. [0189]
(b) A drug-resistance colony formation assay was used to quantitate integration efficiency. The puromycin resistance expression vector pCMV-pur was outfitted with an attB (SEQ ID NO: 4 shown in FIG. 12) or an attP (SEQ ID NO: 5 shown in FIG. 13) sites. Puromycin resistance vectors bearing attB sites were cotransfected with phiC31 integrase or a control vector into DF-1 cells. One day after transfection, puromycin was added. Puromycin resistant colonies were counted 12 days post-transfection. [0190]
In the absence of co-transfected integrase expression, few DF-1 cell colonies were observed after survival selection. When integrase was co-expressed, multiple DF-1 cell colonies were observed, as shown in FIG. 3. Similar to the luciferase expression experiment, the attB sequence (but not the attP sequence) was able to facilitate integration of the plasmid into the genome. FIG. 3 also shows that phiC31 integrase functions at both 37° Celsius and 41° Celsius. Integrase also functions in quail cells using the puromycin resistance assay, as shown in FIG. 4. [0191]
(c) The CMV-pur-attB vector (SEQ ID NO: 4) was also contransfected with an enhanced green fluorescent protein (EGFP) expression vector bearing an attB site (SEQ ID NO: 6 shown in FIG. 14) into DF-1 cells and the phiC31 integrase expression vector CMV-C31int (SEQ ID NO: 1). After puromycin selection for 12 days, the colonies were viewed with UV light to determine the percentage of cells that expressed EGFP. Approximately 20% of puromycin resistant colonies expressed EGFP in all of the cells of the colony, as shown in FIG. 5, indicating that the integrase can mediate multiple integrations per cell. [0192]
(d) PhiC31 integrase promoted the integration of large transgenes into avian cells. A puromycin expression cassette comprising a CMV promoter, puromycin resistance gene, polyadenylation sequence and the attB sequence was inserted into a vector containing a 12.0 kb lysozyme promoter and the human interferon α2b gene (SEQ ID NO: 7 shown in FIG. 15) and into a vector containing a 10.0 kb ovomucoid promoter and the human interferon α2b gene (SEQ ID NO: 8) as shown in FIG. 16. [0193]
DF-1 cells were transfected with donor plasmids of varying lengths bearing a puromycin resistance gene and an attB sequence in the absence or presence of an integrase expression plasmid. Puromycin was added to the culture media to kill those cells which did not contain a stably integrated copy of the puromycin resistance gene. Cells with an integrated gene formed colonies in the presence of puromycin in 7-12 days. The colonies were visualized by staining with methylene blue and the entire 60 mm culture dish was imaged. [0194]
PhiC31 integrase mediated the efficient integration of both vectors as shown in FIG. 7. [0195]

EXAMPLE 2

Cell Culture Methods

DF-1 cells were cultured in DMEM with high glucose, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at 37° Celsius and 5% CO[0196] ₂. A separate population of DF-1 cells was grown at 41° Celsius. These cells were adapted to the higher temperature for one week before they were used for experiments.
Quail QT6 cells were cultured in F10 medium (Gibco) with 5% newborn calf serum, 1% chicken serum heat inactivated (at 55° Celsius for 45 mins), 10 units/ ml penicillin and 10 μg/ml streptomycin at 37° Celsius and 5% CO[0197] ₂.

EXAMPLE 3

Selection and Assay Methods

(a) Puromycin selection assay: About 0.8×10[0198] ⁶DF-1 (chicken) or QT6 (quail) cells were plated in 60 mm dishes. The next day, the cells were transfected as follows:
10 to 50 ng of a donor plasmid and 1 to 10 μg of an Integrase-expressing plasmid DNA were mixed with 150 μl of OptiMEM. 15 μl of DMRIE-C was mixed with 150 μl of OptiMEM in a separate tube, and the mixtures combined and incubated for 15 mins. at room temperature. [0199]
While the liposome/DNA complexes were forming, the cells were washed with OptiMEM and 2.5 ml of OptiMEM was added. After 15 minutes, 300 μl of the DNA-lipid mixture was added dropwise to the 2.5 ml of OptiMEM covering the cell layers. The cells were incubated for 4-5 hours at either 37° Celsius or 41° Celsius, 5% CO[0200] ₂. The transfection mix was replaced with 3 mls of culture media. The next day, puromycin was added to the media at a final concentration of 1 ug/ml, and the media replaced every 2 to 4 days. Puromycin resistant colonies were counted or imaged 10-12 days after the addition of puromycin.
(b) Luciferase assay: Chicken DF-1 or quail QT6 cells (0.8×10[0201] ⁶) were plated in 60 mm dishes. Cells were transfected as described above. The cells from a plate were transferred to a new 100 mm plate when the plate became confluent, typically on day 3-4, and re-passaged every 3-4 days.
At each time point, one-third of the cells from a plate were replated, and one-third were harvested for the luciferase assay. The cells were pelleted in an eppendorf tube and frozen at −70° C. [0202]
The cell pellet was lysed in 200 μl of lysis buffer (25 mM Tris-acetate, pH7.8, 2 mM EDTA, 0.5% Triton X-100, 5% glycerol). Sample (5 μl) was assayed using the Promega BrightGlo reagent system. [0203]
(c) Visualization of EGFP. EGFP expression was visualized with an inverted microscope with FITC illumination [Olympus IX70, 100 W mercury lamp, HQ-FITC Band Pass Emission filter cube, exciter 480/40 nm, emission 535/50 nm, 20X phase contrast objective (total magnification was 2.5×10×20)]. [0204]
(d) Staining of cell colonies: After colonies had formed, typically after 7-12 days of culture in puromycin medium, the cells were fixed in 2% formaldehyde, 0.2% glutaraldehyde for 15 mins, and stained in 0.2% methylene blue for 30 mins. followed by several washes with water. The plates were imaged using a standard CCD camera in visible light. [0205]

EXAMPLE 4

Generation of Genetically Transferred Avian Cells

Avian stage X blastodermal cells are used as the cellular vector for the transgenes. Stage X embryos are collected and the cells dispersed and mixed with plasmid DNA. The transgenes are then introduced to blastodermal cells via electroporation. The cells are immediately injected back into recipient embryos. [0206]
The cells are not cultured for any time period to ensure that they remain capable of contributing to the germline of resulting chimeric embryos. However, because there is no culture step, cells that bear the transgene cannot be identified. Typically, only a small percentage of cells introduced to an embryo will bear a stably integrated transgene (0.01 to 1%). To increase the percentage of cells bearing a transgene, therefore, the transgene vector bears an attB site and is co-electroporated with a vector bearing the CMV promoter driving expression of the phiC31 transgene (CMV-C31int (SEQ ID NO: 1)). The integrase then drives integration of the transgene vector into the nuclear genome of the avian cell and increases the percentage of cells bearing a stable transgene. [0207]
(a) Preparation of avian stage X blastodermal cells: [0208]
i) Collect fertilized eggs from Barred Rock or White leghorn chickens ([0209] Gallus gallus) or quail (Japonica coturnix) within 48 hrs. of laying;
ii) Use 70% ethanol to clean the shells; [0210]
iii) Crack the shells and open the eggs; [0211]
iv) Remove egg whites by transferring yolks to opposite halves of shells, repeating to remove most of the egg whites; [0212]
v) Put egg yolks with embryo discs facing up into a 10 cm petri dish; [0213]
vi) Use an absorbent tissue to gently remove egg white from the embryo discs; [0214]
vii) Place a [0215] Whatman filter paper 1 ring over the embryos;
viii) Use scissors to cut the membranes along the outside edge of the paper ring while gently lifting the ring/embryos with a pair of tweezers; [0216]
ix) Insert the paper ring with the embryos at a 45 degrees angle into a petri dish containing PBS-G solution at room temperature; [0217]
x) After ten embryo discs are collected, gently wash the yolks from the blastoderm discs using a Pasteur pipette under a stereo microscope; [0218]
xi) Cut the discs by a hair ring cutter (a short piece of human hair is bent into a small loop and fastened to the narrow end of a Pasteur pipette with Parafilm); [0219]
xii) Transfer the discs to a 15 ml sterile centrifuge tube on ice; [0220]
xiii) [0221] Place 10 to 15 embryos per tube and allow to settle to the bottom (about 5 mins.);
xiv) Aspirate the supernatant from the tube; [0222]
xv) Add 5 mls of ice-cold PBS without Ca[0223] ⁺⁺ and Mg⁺⁺, and gently pipette 4 to 5 times using a 5 mls pipette;
xvi) Incubate in ice for 5-7 mins. to allow the blastoderms to settle, and aspirate the supernatant; [0224]
xvii) Add 3 mls of ice cold 0.05% trypsin/0.02% ETDA to each tube and gently pipette 3 to 5 times using a 5 ml pipette; [0225]
xviii) Put the tube in ice for 5 mins. and then flick the tube by [0226] finger 40 times. Repeat;
xix) Add 0.5 mls FBS and 3-5 mls BDC medium to each tube and gently pipette 5-7 times using a 5 ml pipette; [0227]
xx) Spin at 500 rpm (RCF 57×g) at 4° Celsius for 5 mins; [0228]
xxi) Remove the supernatant and add 2 mls ice cold BDC medium into each tube; and [0229]
xxii) Resuspend the cells by gently pipetting 20-25 times; and [0230]
xxiii) Determine the cell titer by hemacytometer and ensure that about 95% of all BDCs are single cells, and not clumped. [0231]
(b) Transfection of linearized plasmids into blastodermal cells by small scale electroporation: [0232]
i) Centrifuge the blastodermal cell suspension from step (xxiii) above at RCF 57×g, 4° Celsius, for 5 mins; [0233]
ii) Resuspend cells to a density of 1-3×10[0234] ⁶per ml with PBS without Ca²⁺ and Mg²⁺;
iii) Add linearized DNA, 1-30 μg per 1-3×10[0235] ⁵blastodermal cells in an eppendorf tube at room temperature. Add equimolar molar amounts of the non-linearized transgene plasmid bearing an attB site, and an integrase expression plasmid;
iv) Incubate at room temperature for 10 mins; [0236]
v) Aliquot 100 μl of the DNA-cell mixture to a 0.1 cm cuvette at room temperature; [0237]
vi) Electroporate at 240 V and 25 μFD (or 100 V and 125 μFD for quail cells) using, for example, a Gene Pulser II™ (BIO-RAD). [0238]
vii) Incubate the cuvette at room temperature for 1-10 mins. [0239]
viii) Before the electroporated cells are injected into a recipient embryo, they are transferred to a eppendorf tube at room temperature. The cuvette is washed with 350 μl of media, which is transferred to the eppendorf, spun at room temperature and re-suspended in 0.01-0.3 ml medium; [0240]
ix) Inject 1-10 μl of cell suspension into the subgerminal cavity of an non-irradiated or, preferably, an irradiated (e.g., with 300-900 rads) stage X egg. Shell and shell membrane are removed and, after injection, resealed according to U.S. Pat. No. 6,397,777 incorporated herein by reference in its entirety; and [0241]
x) The egg is then incubated to hatching. [0242]
(c) Blastodermal Cell Culture Medium: [0243]
i) 409.5 mls DMEM with high glucose, L-glutamine, sodium pyruvate, pyridoxine hydrochloride; [0244]
ii) 5 mls Men non-essential amino acids solution, 10 mM; [0245]
iii) 5 mls Penicillin-streptomycin 5000 U/ml each; [0246]
iv) 5 mls L-glutamine, 200 mM; [0247]
v) 75 mls fetal bovine serum; and [0248]
vi) 0.5 mls β-mercaptoethanol, 11.2 mM. [0249]

EXAMPLE 5

Transfection of Stage X Embryos With attB Plasmids

(a) DNA-PEI. Twenty-five μg of a phage phiC31 integrase expression plasmid (pCMV-int), and 25 μg of a luciferase-expressing plasmid (pβ-actin-GFP-attB) are combined in 200 μl of 28 mM Hepes (pH 7.4). The DNA/Hepes is mixed with an equal volume of PEI which has been diluted 10-fold with water. The DNA/Hepes/PEI is incubated at room temperature for 15 mins Three to seven μl of the complex are injected into the subgerminal cavity of windowed stage X white leghorn eggs which are then sealed and incubated as described in U.S. Pat. No. 6,397,777. The complexes will also be incubated with blastodermal cells isolated from stage X embryos which are subsequently injected into the subgerminal cavity of windowed irradiated stage X white leghorn eggs. Injected eggs are sealed and incubated as described above. [0250]
(b) Adenovirus-PEI: [0251]
Two μg of a phage phiC31 integrase expression plasmid (pCMV-int), 2 μg of a GFP expressing plasmid (pβ-actin-GFP-attB) and 2 μg of a luciferase expressing plasmid (pGLB) were incubated with 1.2 μl of JetPEI™ in 50 μl of 20 mM Hepes buffer (pH7.4). After 10 mins at 25° C., 3×10[0252] ⁹adenovirus particles (Ad5-Null, Qbiogene) were added and the incubation continued for an additional 10 mins. Embryos are transfected in ovo or ex ovo as described above.

EXAMPLE 6

Stage I Cytoplasmic Injection

Production of transgenic chickens by cytoplasmic DNA injection using DNA injection directly into the germinal disk as described in Sang et al., [0253] Mol. Reprod. Dev., 1: 98-106 (1989); Love et al., Biotechnology, 12: 60-63 (1994) incorporated herein by reference in their entireties.
In the method of the present invention, fertilized ova, and preferably stage I embryos, are isolated from euthanized hens 45 mins. to 4 hrs. after oviposition of the previous egg. Alternatively, eggs were isolated from hens whose oviducts have been fistulated according to the techniques of Gilbert & Wood-Gush, [0254] J. Reprod. Fertil., 5: 451-453 (1963) and Pancer et al., Br. Poult. Sci., 30: 953-7 (1989) incorporated herein in their entireties.
An isolated ovum was placed in dish with the germinal disk upwards. Ringer's buffer medium was then added to prevent drying of the ovum. Any suitable microinjection assembly and methods for microinjecting and reimplanting avian eggs are useful in the method of cytoplasmic injection of the present invention. A particularly suitable apparatus and method for use in the present invention is described in U.S. patent application Ser. No.: 09/919,143 (“the '143 Application) and incorporated herein by reference in its entirety. The avian microinjection system described in the '143 Application allowed the loading of a DNA solution into a micropipette, followed by prompt positioning of the germinal disk under the microscope and guided injection of the DNA solution into the germinal disk. Injected embryos could then be surgically transferred to a recipient hen as described, for example, in Olsen & Neher, [0255] J. Exp. Zool., 109: 355-66 (1948) and Tanaka et al., J. Reprod. Fertil., 100: 447-449 (1994). The embryo was allowed to proceed through the natural in vivo cycle of albumin deposition and hard-shell formation. The transgenic embryo is then laid as a hard-shell egg which was incubated until hatching of the chick. Preferably, injected embryos were surgically transferred to recipient hens via the ovum transfer method of Christmann et al. in PCT/US01/26723, the contents of which are incorporated by reference in its entirety, and hard shell eggs were incubated and hatched.
Approximately 25 nl of DNA solution with either integrase mRNA or protein were injected into a germinal disc of stage I White Leghorn embryos obtained 90 minutes after oviposition of the preceding egg. Typically the concentration of integrase mRNA used was 100 ng/μl, and the concentration of integrase protein was 66 ng/μl. [0256]
To synthesize the integrase mRNA, a plasmid template encoding the integrase protein was linearized at the 3′ end of the transcription unit. mRNA was synthesized, capped and a polyadenine tract added using the mMESSAGE mMACHINE T7 Ultra Kit™ (Ambion, Austin, Tex.). The mRNA was purified by extraction with phenol and chloroform and precipitiated with isopropanol. The integrase protein was expressed in [0257] E. coli and purified as described by Thorpe et al., Mol. Microbiol., 38: 232-241 (2000).
A plasmid encoding for the integrase protein is transfected into the target cells. However, since the early avian embryo transcriptionally silent until it reaches about 22,000 cells, injection of the integrase mRNA or protein was expected to result in better rates of transgenesis, as shown in the Table 1 below. [0258]
The chicks produced by this procedure were screened for the presence of the injected transgene using a high throughput PCR-based screening procedure as described in Harvey et al., [0259] Nature Biotech., 20: 396-399 (2002).

TABLE 1

Summary of cytoplasmic injection results using different integrase

strategies

Experi-

mental Ovum Hard shells Chicks Transgenic

group transfers produced (%) hatched (%)* chicks (%)^‡

No Integrase 5164 3634 (70%) 500 (14%) 58 (11.6%)

Integrase 1109 833 (75%) 115 (13.8%) 19 (16.5%)

mRNA

Integrase 374 264 47 (17.8%) 16 (34%)

protein (70.6%)

EXAMPLE 7

Characterization of phiC31 Integrase-Mediated Integration Sites in the Chicken Genome

To characterize phiC31 -mediated integration into the chicken genome, a plasmid rescue method was used to isolate integrated plasmids from transfected and selected chicken fibroblasts. Plasmid pCR-XL-TOPO-CMV-pur-attB (SEQ ID NO: 10, shown in FIG. 18) does not have BamH I or Bgl II restriction sites. Genomic DNA from cells transformed with pCR-XL-TOPO-CMV-pur-attB was cut with BamH I or Bgl II (either or both of which would cut in the flanking genomic regions) and religated so that the genomic DNA surrounding the integrated plasmid would be captured into the circularized plasmid. The flanking DNA of a number of plasmids were then sequenced. [0260]
DF-1 cells (chicken fibroblasts), 4×10[0261] ⁵were transfected with 50 ng of pCR-XL-TOPO-CMV-pur-attB and 1 μg of pCMV-int. The following day, the culture medium was replaced with fresh media supplemented with 1 μg/ml puromycin. After 10 days of selection, several hundred puromycin-resistant colonies were evident. These were harvested by trypsinzation, pooled, replated on 10 cm plates and grown to confluence. DNA was then extracted.
Isolated DNA was digested with BamH I and Bgl II for 2-3 hrs, extracted with phenol:chloroform:isoamyl alcohol chloroform:isoamyl alcohol and ethanol precipitated. T4 DNA ligase was added and the reaction incubated for 1 hr at room temperature, extracted with phenol:chloroform:isoamyl alcohol and chloroform:isoamyl alcohol, and precipitated with ethanol. 5 μl of the DNA suspended in 10 μl of water was electroporated into 25 μl of Genehogs™ (Invitrogen) in an 0.1 cm cuvette using a GenePulser II (Biorad) set at 1.6 kV, 100 ohms, 25 uF and plated on Luria Broth (LB) plates with 5 μg/ml phleomycin (or 25 μg/ml zeocin) and 20 μg/ml kanamycin. Approximately 100 individual colonies were cultured, the plasmids extracted by standard miniprep techniques and digested with Xba I to identify clones with unique restriction fragments. [0262]
Thirty two plasmids were sequenced with the primer attB-for (5′-TACCGTCGACGATGTAGGTCACGGTC-3′) (SEQ ID NO: 12) which allows sequencing across the crossover site of attB and into the flanking genomic sequence. All of plasmids sequenced had novel sequences inserted into the crossover site of attB, indicating that the clones were derived from plasmid that had integrated into the chicken genome via phiC31 integrase-mediated recombination. [0263]
The sequences were compared with sequences at GenBank using Basic Local Alignment Search Tool (BLAST). Most of the clones harbored sequences homologous to Gallus genomic sequences in the TRACE database. [0264]

EXAMPLE 8

Insertion of a Wild-Type attP Site Into the Avian Genome Augments Integrase-Mediated Integration and Transgenesis

The chicken B-cell line DT40 cells (Buerstedde et al., [0265] E.M.B.O. J., 9: 921-927 (1990)) are useful for studying DNA integration and recombination processes (Buerstedde & Takeda, Cell, 67:179-88 (1991)). DT40 cells were engineered to harbor a wild-type attP site isolated from the Streptomyces phage phiC31. Two independent cell lines were created by transfection of a linearized plasmid bearing an attP site linked to a CMV promoter driving the resistance gene to G418 (DT40-NLB-attP) or bearing an attP site linked to a CMV promoter driving the resistance gene for puromycin (DT40-pur-attP). The transfected cells were cultured in the presence of G418 or puromycin to enrich for cells bearing an attP sequence stably integrated into the genome.
A super-coiled luciferase vector bearing an attB (SEQ ID NO: 2 shown in FIG. 10) was co-transfected, together with an integrase expression vector CMV-C31int (SEQ ID NO: 1) or a control, non-integrase expressing vector (CMV-BL) into wild-type DT40 cells and the stably transformed lines DT40-NLB-attP and DT40-pur-attP. [0266]
Cells were passaged at 5, 7 and 14 days post-transfection and about one third of the cells were harvested and assayed for luciferase. The expression of luciferase was plotted as a percentage of the expression measured 5 days after transfection. As can be seen in FIG. 21, in the absence of integrase, or in the presence of integrase but in the DT40 cells lacking an inserted wild-type attP site, luciferase expression from a vector bearing attB progressively decreased to very low levels. However, luciferase levels were persistent when the luciferase vector bearing attB was co-transfected with the integrase expression vector into the attP bearing cell lines DT40-NLB-attP and DT40-pur-attP. Inclusion of an attP sequence in the avian genome augments the level of integration efficiency beyond that afforded by the utilization of endogenous pseudo-attP sites. [0267]

EXAMPLE 9

Generation of attP Transgenic Cell Line and Birds Using an NLB Vector

The NLB-attP retroviral vector can be injected into stage X chicken embryos laid by pathogen-free hens. A small hole is drilled into the egg shell of a freshly laid egg, the shell membrane cut away and the embryo visualized by eye. With a drawn needle attached to a syringe, 1 to 10 μl of concentrated retrovirus, approximately 2.5×10[0268] ⁵IU, is injected into the subgerminal cavity of the embryo. The egg shell is resealed with a hot glue gun. Suitable methods for the manipulation of avian eggs, including opening and resealing hard shell eggs are described in U.S. patent Ser. Nos.: 5,897,998 and 6,397,777 which are herein incorporated by reference in their entireties.
Typically, 25% of embryos hatch 21 days later. The chicks are raised to sexual maturity and semen samples are taken. Birds that have a significant level of the transgene in sperm DNA will be identified, typically by a PCR-based assay. Ten to 25% of the hatched roosters will be able to give rise to G1 transgenic offspring, 1 to 20% of which may be transgenic. DNA extracted from the blood of G1 offspring is analyzed by PCR and Southern analysis to confirm the presence of the intact transgene. Several lines of transgenic roosters, each with a unique site of attP integration, are then bred to non-transgenic hens, giving 50% of G2 transgenic offspring. Transgenic G2 hens and roosters from the same line can be bred to produce G3 offspring homozygous for the transgene. Homozygous offspring will be distinguished from hemizygous offspring by quantitative PCR. The same procedure can be used to integrate an attB or attP site into transgenic birds. [0269]

EXAMPLE 10

Expression of Immunoglobulin Chain Polypeptides by Transgenic Chickens

Bacterial artificial chromosomes (BACs) containing a 70 kbp segment of the chicken ovomucoid gene with the light and heavy chain cDNAs for a human monoclonal antibody inserted along with an internal ribosome entry site into the 3′ untranslated region of the ovomucoid gene were equipped with the attB sequence. The heavy and light chain cDNAs were inserted into separate ovomucoid BACs such that expression of an intact monoclonal antibody requires the presence of both BACs in the nucleus. [0270]
Several hens produced by coinjection of the attB-bearing ovomucoid BACs and integrase-encoding mRNA into stage I embryos produced intact monoclonal antibodies in their egg white. One hen, which had a high level of the light chain ovomucoid BAC in her blood DNA as determined by quantitative PCR particularly expressed the light chain portion of the monoclonal antibody in the egg white at a concentration of 350 nanograms per ml, or approximately 12 μg per egg. [0271]
1 12 1 6230 DNA Plasmid pCMV-31int 1 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 60 ttacgccagc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg 120 atcgatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 180 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 240 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 300 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat 360 gatcatgaac agactgtgag gactgagggg cctgaaatga gccttgggac tgtgaatcta 420 aaatacacaa acaattagaa tcactagctc ctgtgtataa tattttcata aatcatactc 480 agtaagcaaa actctcaagc agcaagcata tgcagctagt ttaacacatt atacacttaa 540 aaattttata tttaccttag agctttaaat ctctgtaggt agtttgtcca attatgtcac 600 accacagaag taaggttcct tcacaaagat cccaagctag cttataatac gactcactat 660 agggagagag ctatgacgtc gcatgcacgc gtaagcttgg gcccctcgag ggatccgggt 720 gtctcgctac gccgctacgt cttccgtgcc gtcctgggcg tcgtcttcgt cgtcgtcggt 780 cggcggcttc gcccacgtga tcgaagcgcg cttctcgatg ggcgttccct gccccctgcc 840 cgtagtcgac ttcgtgacaa cgatcttgtc tacgaagagc ccgacgaaca cgcgcttgtc 900 gtctactgac gcgcgccccc accacgactt agggccggtc gggtcagcgt cggcgtcttc 960 ggggaaccat tggtcaaggg gaagcttcgg ggcttcggcg gcttcaagtt cggcaagccg 1020 ctcttccgcc ccttgctgcc ggagcgtcag cgctgcctgt tgcttccgga agtgcttcct 1080 gccaacgggt ccgtcgtacg cgcctgccgc gcggtcttcg tacagctctt caagggcgtt 1140 cagggcgtcg gcgcgctccg caacaaggtt cgcccgttcg ccgctcttct caggcgcctc 1200 agtgagcttg ccgaagcgtc gggcggcttc ccacagaagc gccaacgtct cttcgtcgcc 1260 ttcggcgtgc ctgatcttgt tgaagatgcg ttccgcaacg aacttgtcga gtgccgccat 1320 gctgacgttg cacgtgcctt cgtgctgccc aggtgcggac gggtcgacca ccttccggcg 1380 acggcagcgg taagagtcct tgatcgattc ttccccgcgc ttcgaagtca tgacggcgcc 1440 acactcgcag tacagcttgt ccatggcgga cagaatggct tgcccccggg aaagcccctt 1500 gccgcgcccc ctgccgtcca accacgcctg aagctcatac cactcagcgg gctcgatgat 1560 cggtccgcaa tcaagctcga ccggccggag cgtgatcggg tcgcgctgaa tgcggtaacc 1620 ctcaatcttc gtggtcggcg tgccgtccgg cttcttcttg tagatcacct cagcggcgaa 1680 gcccgcaata cgcgggtccc gaaggattcg cataacggtt gccgggtccc aggcgcttga 1740 agcggtcttc ttcccaatcg tctcgccccg ggtcggcacg gcgtcagcgt ccatgcgctt 1800 acaaagcccc gtgatgctgc ccgggtgaat ggcggcttga ctgcccggct tgaagggaag 1860 gtgtttgtgc gtcttgatct cacgccacca ccaccggatt acgtcgggct cgaactcgaa 1920 gggtccggta aggggagtgg tcgagtgcgc aagcttgttg atgacgacat tgaccattcg 1980 gccgttgcgc gtgatctcct tcgtctccga aacaagctcg aagccgtaag gcgccttccc 2040 gccgacgtac ccgcccaatt cgcgctgaag gttcttcgtg tcgagaatct tcgccgactt 2100 cagcgaagat tctttgtgcg acgcgtcgag ccgcataatc aggtgaatca ggtccatgac 2160 gtttccctgc cggaagacgc cttcctgagt ggaaacaatc gtcacgccca gggcgagcaa 2220 ttccgagaca atcggaatcg cgtccatgac cttcaggcgc gagaagcgcg acacgtcata 2280 gacaatgatc atgttgagcc gcccggcgcg gcattcgttc aggatgcgtt cgaactccgg 2340 gcgctccgcc gtcccgaacg ccgacgtgcc cggcgcttcg ctgaaatgcc cgacgaacct 2400 gaaccggccc ccgtcgcgct cgacttcgcg ctgaaggtcg gccgccttgt cttcgttggc 2460 gctacgctgt gtcgctgggc ttgctgcgct cgaattctcg cgctcgcgcg actgacggtc 2520 gtaagcaccc gcgtacgtgt ccaccccggt cacaacccct tgtgtcatgt cggcgaccct 2580 acgactagtg agctcgtcga cccgggaatt ccggaccggt acctgcaggc gtaccttcta 2640 tagtgtcacc taaatagctt tttgcaaaag cctaggctag agtccggagg ctggatcggt 2700 cccggtgtct tctatggagg tcaaaacagc gtggatggcg tctccaggcg atctgacggt 2760 tcactaaacg agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg 2820 tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca 2880 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc 2940 cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt 3000 agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg 3060 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta 3120 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta 3180 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg 3240 gtcagccagg cgggccattt accgtaagtt atgtaacgac ctgcacgatg ctgtttcctg 3300 tgtgaaattg ttatccgctc acaattccac acattatacg agccggaagc tataaagtgt 3360 aaagcctggg gtgcctaatg agtgaaaggg cctcgtatac gcctattttt ataggttaat 3420 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga 3480 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 3540 ccctgataaa tgcttcaata atattgaaaa acgcgcgaat tgcaagctct gcattaatga 3600 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 3660 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 3720 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 3780 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 3840 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 3900 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 3960 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 4020 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4080 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4140 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4200 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4260 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4320 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4380 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 4440 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgcc ataacttcgt 4500 atagcataca ttatacgaag ttatggcatg agattatcaa aaaggatctt cacctagatc 4560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 4620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 4680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 4740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 4800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 4860 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 4920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 4980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 5280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 5340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 5400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 5460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 5520 cagggttatt gtctcatgcc aggggtgggc acacatattt gataccagcg atccctacac 5580 agcacataat tcaatgcgac ttccctctat cgcacatctt agacctttat tctccctcca 5640 gcacacatcg aagctgccga gcaagccgtt ctcaccagtc caagacctgg catgagcgga 5700 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5760 aaagtgccac ctgaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt 5820 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 5880 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 5940 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 6000 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 6060 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 6120 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 6180 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 6230 2 5982 DNA Plasmid pCMV-luc-attB 2 ctctatcgat aggtaccgag ctcttacgcg tgctagccct cgagcaggat ctatacattg 60 aatcaatatt ggcaattagc catattagtc attggttata tagcataaat caatattggc 120 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt 180 ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg 240 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc 300 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 360 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 420 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat 480 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact 540 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac 600 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac 660 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac 720 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga 780 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat 840 agaagacacc gggaccgatc cagcctcccc tcgaagctcg actctagggg ctcgagatct 900 gcgatctaag taagcttggc attccggtac tgttggtaaa gccaccatgg aagacgccaa 960 aaacataaag aaaggcccgg cgccattcta tccgctggaa gatggaaccg ctggagagca 1020 actgcataag gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc 1080 acatatcgag gtggacatca cttacgctga gtacttcgaa atgtccgttc ggttggcaga 1140 agctatgaaa cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc 1200 tcttcaattc tttatgccgg tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc 1260 gaacgacatt tataatgaac gtgaattgct caacagtatg ggcatttcgc agcctaccgt 1320 ggtgttcgtt tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat 1380 catccaaaaa attattatca tggattctaa aacggattac cagggatttc agtcgatgta 1440 cacgttcgtc acatctcatc tacctcccgg ttttaatgaa tacgattttg tgccagagtc 1500 cttcgatagg gacaagacaa ttgcactgat catgaactcc tctggatcta ctggtctgcc 1560 taaaggtgtc gctctgcctc atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc 1620 tatttttggc aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca 1680 cggttttgga atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat 1740 gtatagattt gaagaagagc tgtttctgag gagccttcag gattacaaga ttcaaagtgc 1800 gctgctggtg ccaaccctat tctccttctt cgccaaaagc actctgattg acaaatacga 1860 tttatctaat ttacacgaaa ttgcttctgg tggcgctccc ctctctaagg aagtcgggga 1920 agcggttgcc aagaggttcc atctgccagg tatcaggcaa ggatatgggc tcactgagac 1980 tacatcagct attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt 2040 tgttccattt tttgaagcga aggttgtgga tctggatacc gggaaaacgc tgggcgttaa 2100 tcaaagaggc gaactgtgtg tgagaggtcc tatgattatg tccggttatg taaacaatcc 2160 ggaagcgacc aacgccttga ttgacaagga tggatggcta cattctggag acatagctta 2220 ctgggacgaa gacgaacact tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa 2280 aggctatcag gtggctcccg ctgaattgga atccatcttg ctccaacacc ccaacatctt 2340 cgacgcaggt gtcgcaggtc ttcccgacga tgacgccggt gaacttcccg ccgccgttgt 2400 tgttttggag cacggaaaga cgatgacgga aaaagagatc gtggattacg tcgccagtca 2460 agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag taccgaaagg 2520 tcttaccgga aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg 2580 cggaaagatc gccgtgtaat tctagagtcg gggcggccgg ccgcttcgag cagacatgat 2640 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 2700 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 2760 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 2820 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcaat tcggcttcag 2880 gtaccgtcga cgatgtaggt cacggtctcg aagccgcggt gcgggtgcca gggcgtgccc 2940 ttgggctccc cgggcgcgta ctccacctca cccatctggt ccatcatgat gaacgggtcg 3000 aggtggcggt agttgatccc ggcgaacgcg cggcgcaccg ggaagccctc gccctcgaaa 3060 ccgctgggcg cggtggtcac ggtgagcacg ggacgtgcga cggcgtcggc gggtgcggat 3120 acgcggggca gcgtcagcgg gttctcgacg gtcacggcgg gcatgtcgac agccgaattg 3180 atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg 3240 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag 3300 gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 3360 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3420 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3480 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3540 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3600 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3660 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt 3720 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3780 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 3840 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 3900 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 3960 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4020 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 4080 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 4140 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 4200 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 4260 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 4320 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 4380 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 4440 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 4500 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 4560 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 4620 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 4680 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 4740 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 4800 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 4860 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 4920 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 4980 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 5040 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 5100 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 5160 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 5220 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg 5280 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5340 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5400 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5460 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5520 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5580 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 5640 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 5700 ttcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 5760 gctattacgc cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact 5820 tggagcggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 5880 gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca 5940 aaataggctg tccccagtgc aagtgcaggt gccagaacat tt 5982 3 5924 DNA Plasmid pCMV-luc-attP 3 ctctatcgat aggtaccgag ctcttacgcg tgctagccct cgagcaggat ctatacattg 60 aatcaatatt ggcaattagc catattagtc attggttata tagcataaat caatattggc 120 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt 180 ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg 240 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc 300 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 360 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 420 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat 480 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact 540 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac 600 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac 660 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac 720 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga 780 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat 840 agaagacacc gggaccgatc cagcctcccc tcgaagctcg actctagggg ctcgagatct 900 gcgatctaag taagcttggc attccggtac tgttggtaaa gccaccatgg aagacgccaa 960 aaacataaag aaaggcccgg cgccattcta tccgctggaa gatggaaccg ctggagagca 1020 actgcataag gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc 1080 acatatcgag gtggacatca cttacgctga gtacttcgaa atgtccgttc ggttggcaga 1140 agctatgaaa cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc 1200 tcttcaattc tttatgccgg tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc 1260 gaacgacatt tataatgaac gtgaattgct caacagtatg ggcatttcgc agcctaccgt 1320 ggtgttcgtt tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat 1380 catccaaaaa attattatca tggattctaa aacggattac cagggatttc agtcgatgta 1440 cacgttcgtc acatctcatc tacctcccgg ttttaatgaa tacgattttg tgccagagtc 1500 cttcgatagg gacaagacaa ttgcactgat catgaactcc tctggatcta ctggtctgcc 1560 taaaggtgtc gctctgcctc atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc 1620 tatttttggc aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca 1680 cggttttgga atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat 1740 gtatagattt gaagaagagc tgtttctgag gagccttcag gattacaaga ttcaaagtgc 1800 gctgctggtg ccaaccctat tctccttctt cgccaaaagc actctgattg acaaatacga 1860 tttatctaat ttacacgaaa ttgcttctgg tggcgctccc ctctctaagg aagtcgggga 1920 agcggttgcc aagaggttcc atctgccagg tatcaggcaa ggatatgggc tcactgagac 1980 tacatcagct attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt 2040 tgttccattt tttgaagcga aggttgtgga tctggatacc gggaaaacgc tgggcgttaa 2100 tcaaagaggc gaactgtgtg tgagaggtcc tatgattatg tccggttatg taaacaatcc 2160 ggaagcgacc aacgccttga ttgacaagga tggatggcta cattctggag acatagctta 2220 ctgggacgaa gacgaacact tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa 2280 aggctatcag gtggctcccg ctgaattgga atccatcttg ctccaacacc ccaacatctt 2340 cgacgcaggt gtcgcaggtc ttcccgacga tgacgccggt gaacttcccg ccgccgttgt 2400 tgttttggag cacggaaaga cgatgacgga aaaagagatc gtggattacg tcgccagtca 2460 agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag taccgaaagg 2520 tcttaccgga aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg 2580 cggaaagatc gccgtgtaat tctagagtcg gggcggccgg ccgcttcgag cagacatgat 2640 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 2700 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 2760 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 2820 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcaat tcggcttcga 2880 ctagtactga cggacacacc gaagccccgg cggcaaccct cagcggatgc cccggggctt 2940 cacgttttcc caggtcagaa gcggttttcg ggagtagtgc cccaactggg gtaacctttg 3000 agttctctca gttgggggcg tagggtcgcc gacatgacac aaggggttgt gaccggggtg 3060 gacacgtacg cgggtgctta cgaccgtcag tcgcgcgagc gcgactagta caagccgaat 3120 tgatccgtcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc 3180 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac 3240 aggtgccggc agcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3300 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3360 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3420 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3480 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 3540 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 3600 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 3660 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 3720 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 3780 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 3840 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 3900 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 3960 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4020 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4080 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 4140 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 4200 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 4260 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 4320 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 4380 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 4440 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 4500 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 4560 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 4620 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 4680 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 4740 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 4800 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 4860 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 4920 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 4980 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 5040 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 5100 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 5160 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg 5220 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 5280 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 5340 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 5400 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 5460 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 5520 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 5580 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 5640 atttcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 5700 tcgctattac gccagcccaa gctaccatga taagtaagta atattaaggt acgggaggta 5760 cttggagcgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt 5820 gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag 5880 caaaataggc tgtccccagt gcaagtgcag gtgccagaac attt 5924 4 5101 DNA Plasmid pCMV-pur-attB 4 ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg 60 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 120 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 180 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 240 caaatgtggt aaaatcgata aggatcaatt cggcttcagg taccgtcgac gatgtaggtc 300 acggtctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac 360 tccacctcac ccatctggtc catcatgatg aacgggtcga ggtggcggta gttgatcccg 420 gcgaacgcgc ggcgcaccgg gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg 480 gtgagcacgg gacgtgcgac ggcgtcggcg ggtgcggata cgcggggcag cgtcagcggg 540 ttctcgacgg tcacggcggg catgtcgaca gccgaattga tccgtcgacc gatgcccttg 600 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 660 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc 720 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 780 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 840 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 900 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 960 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1020 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1080 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1140 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1200 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 1260 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 1320 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 1380 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 1440 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 1500 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1560 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1620 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 1680 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 1740 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 1800 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 1860 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 1920 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 1980 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 2040 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 2100 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 2160 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 2220 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 2280 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 2340 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 2400 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 2460 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 2520 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 2580 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 2640 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 2700 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 2760 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 2820 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 2880 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 2940 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 3000 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 3060 atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct 3120 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct 3180 accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata 3240 tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac 3300 gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 3360 agtgcaggtg ccagaacatt tctctatcga taggtaccga gctcttacgc gtgctagccc 3420 tcgagcagga tctatacatt gaatcaatat tggcaattag ccatattagt cattggttat 3480 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg 3540 tacatttata ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt 3600 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 3660 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 3720 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 3780 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 3840 ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 3900 accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 3960 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 4020 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 4080 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 4140 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 4200 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctc 4260 gactctaggg gctcgagatc tgcgatctaa gtaagcttgc atgcctgcag gtcggccgcc 4320 acgaccggtg ccgccaccat cccctgaccc acgcccctga cccctcacaa ggagacgacc 4380 ttccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc 4440 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc 4500 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct 4560 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc 4620 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag 4680 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa 4740 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct 4800 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt 4860 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac 4920 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc 4980 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatggctc 5040 cgaccgaagc cgacccgggc ggccccgccg accccgcacc cgcccccgag gcccaccgac 5100 t 5101 5 5043 DNA Plasmid pCMV-pur-attP 5 ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg 60 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 120 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 180 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 240 caaatgtggt aaaatcgata aggatcaatt cggcttcgac tagtactgac ggacacaccg 300 aagccccggc ggcaaccctc agcggatgcc ccggggcttc acgttttccc aggtcagaag 360 cggttttcgg gagtagtgcc ccaactgggg taacctttga gttctctcag ttgggggcgt 420 agggtcgccg acatgacaca aggggttgtg accggggtgg acacgtacgc gggtgcttac 480 gaccgtcagt cgcgcgagcg cgactagtac aagccgaatt gatccgtcga ccgatgccct 540 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg 600 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc 660 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 720 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 780 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 840 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 900 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 960 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1020 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1080 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1140 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1200 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1260 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1320 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1380 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1440 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1500 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1560 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1620 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1680 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1740 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1800 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1860 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1920 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1980 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2040 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2100 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2160 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 2220 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 2280 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2340 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2400 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2460 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2520 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 2580 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 2640 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 2700 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 2760 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 2820 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2880 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 2940 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3000 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcccattc gccattcagg 3060 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag 3120 ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa 3180 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat 3240 acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg 3300 caagtgcagg tgccagaaca tttctctatc gataggtacc gagctcttac gcgtgctagc 3360 cctcgagcag gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt 3420 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata 3480 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta 3540 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 3600 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 3660 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 3720 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 3780 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 3840 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 3900 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 3960 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 4020 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 4080 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc 4140 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc ccctcgaagc 4200 tcgactctag gggctcgaga tctgcgatct aagtaagctt gcatgcctgc aggtcggccg 4260 ccacgaccgg tgccgccacc atcccctgac ccacgcccct gacccctcac aaggagacga 4320 ccttccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg 4380 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac 4440 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg 4500 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg 4560 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg 4620 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc 4680 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt 4740 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc 4800 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc 4860 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt 4920 gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc 4980 tccgaccgaa gccgacccgg gcggccccgc cgaccccgca cccgcccccg aggcccaccg 5040 act 5043 6 5041 DNA Plasmid pCMV-EGFP-attB 6 ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg 60 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 120 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 180 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 240 caaatgtggt aaaatcgata aggatcaatt cggcttcagg taccgtcgac gatgtaggtc 300 acggtctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac 360 tccacctcac ccatctggtc catcatgatg aacgggtcga ggtggcggta gttgatcccg 420 gcgaacgcgc ggcgcaccgg gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg 480 gtgagcacgg gacgtgcgac ggcgtcggcg ggtgcggata cgcggggcag cgtcagcggg 540 ttctcgacgg tcacggcggg catgtcgaca gccgaattga tccgtcgacc gatgcccttg 600 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 660 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc 720 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 780 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 840 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 900 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 960 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1020 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1080 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1140 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1200 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 1260 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 1320 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 1380 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 1440 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 1500 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1560 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1620 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 1680 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 1740 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 1800 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 1860 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 1920 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 1980 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 2040 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 2100 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 2160 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 2220 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 2280 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 2340 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 2400 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 2460 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 2520 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 2580 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 2640 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 2700 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 2760 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 2820 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 2880 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 2940 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 3000 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 3060 atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct 3120 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct 3180 accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata 3240 tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac 3300 gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 3360 agtgcaggtg ccagaacatt tctctatcga taggtaccga gctcttacgc gtgctagccc 3420 tcgagcagga tctatacatt gaatcaatat tggcaattag ccatattagt cattggttat 3480 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg 3540 tacatttata ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt 3600 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 3660 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 3720 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 3780 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 3840 ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 3900 accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 3960 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 4020 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 4080 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 4140 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 4200 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctc 4260 gactctaggg gctcgagatc cccgggtacc ggtcgccacc atggtgagca agggcgagga 4320 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 4380 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 4440 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 4500 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 4560 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 4620 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 4680 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 4740 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 4800 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 4860 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 4920 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 4980 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgct cgagcatgca 5040 t 5041 7 18116 DNA Plasmid p12.0lys-LSPIPNMM-CMV-pur-attB 7 gggctgcagg aattcgattg ccgccttctt tgatattcac tctgttgtat ttcatctctt 60 cttgccgatg aaaggatata acagtctgta taacagtctg tgaggaaata cttggtattt 120 cttctgatca gtgtttttat aagtaatgtt gaatattgga taaggctgtg tgtcctttgt 180 cttgggagac aaagcccaca gcaggtggtg gttggggtgg tggcagctca gtgacaggag 240 aggttttttt gcctgttttt tttttttttt ttttttttaa gtaaggtgtt cttttttctt 300 agtaaatttt ctactggact gtatgttttg acaggtcaga aacatttctt caaaagaaga 360 accttttgga aactgtacag cccttttctt tcattccctt tttgctttct gtgccaatgc 420 ctttggttct gattgcatta tggaaaacgt tgatcggaac ttgaggtttt tatttatagt 480 gtggcttgaa agcttggata gctgttgtta cacgagatac cttattaagt ttaggccagc 540 ttgatgcttt attttttccc tttgaagtag tgagcgttct ctggtttttt tcctttgaaa 600 ctggtgaggc ttagattttt ctaatgggat tttttacctg atgatctagt tgcataccca 660 aatgcttgta aatgttttcc tagttaacat gttgataact tcggatttac atgttgtata 720 tacttgtcat ctgtgtttct agtaaaaata tatggcattt atagaaatac gtaattcctg 780 atttcctttt tttttatctc tatgctctgt gtgtacaggt caaacagact tcactcctat 840 ttttatttat agaattttat atgcagtctg tcgttggttc ttgtgttgta aggatacagc 900 cttaaatttc ctagagcgat gctcagtaag gcgggttgtc acatgggttc aaatgtaaaa 960 cgggcacgtt tggctgctgc cttcccgaga tccaggacac taaactgctt ctgcactgag 1020 gtataaatcg cttcagatcc cagggaagtg cagatccacg tgcatattct taaagaagaa 1080 tgaatacttt ctaaaatatt ttggcatagg aagcaagctg catggatttg tttgggactt 1140 aaattatttt ggtaacggag tgcataggtt ttaaacacag ttgcagcatg ctaacgagtc 1200 acagcgttta tgcagaagtg atgcctggat gcctgttgca gctgtttacg gcactgcctt 1260 gcagtgagca ttgcagatag gggtggggtg ctttgtgtcg tgttcccaca cgctgccaca 1320 cagccacctc ccggaacaca tctcacctgc tgggtacttt tcaaaccatc ttagcagtag 1380 tagatgagtt actatgaaac agagaagttc ctcagttgga tattctcatg ggatgtcttt 1440 tttcccatgt tgggcaaagt atgataaagc atctctattt gtaaattatg cacttgttag 1500 ttcctgaatc ctttctatag caccacttat tgcagcaggt gtaggctctg gtgtggcctg 1560 tgtctgtgct tcaatctttt aaagcttctt tggaaataca ctgacttgat tgaagtctct 1620 tgaagatagt aaacagtact tacctttgat cccaatgaaa tcgagcattt cagttgtaaa 1680 agaattccgc ctattcatac catgtaatgt aattttacac ccccagtgct gacactttgg 1740 aatatattca agtaatagac tttggcctca ccctcttgtg tactgtattt tgtaatagaa 1800 aatattttaa actgtgcata tgattattac attatgaaag agacattctg ctgatcttca 1860 aatgtaagaa aatgaggagt gcgtgtgctt ttataaatac aagtgattgc aaattagtgc 1920 aggtgtcctt aaaaaaaaaa aaaaaaagta atataaaaag gaccaggtgt tttacaagtg 1980 aaatacattc ctatttggta aacagttaca tttttatgaa gattaccagc gctgctgact 2040 ttctaaacat aaggctgtat tgtcttcctg taccattgca tttcctcatt cccaatttgc 2100 acaaggatgt ctgggtaaac tattcaagaa atggctttga aatacagcat gggagcttgt 2160 ctgagttgga atgcagagtt gcactgcaaa atgtcaggaa atggatgtct ctcagaatgc 2220 ccaactccaa aggattttat atgtgtatat agtaagcagt ttcctgattc cagcaggcca 2280 aagagtctgc tgaatgttgt gttgccggag acctgtattt ctcaacaagg taagatggta 2340 tcctagcaac tgcggatttt aatacatttt cagcagaagt acttagttaa tctctacctt 2400 tagggatcgt ttcatcattt ttagatgtta tacttgaaat actgcataac ttttagcttt 2460 catgggttcc tttttttcag cctttaggag actgttaagc aatttgctgt ccaacttttg 2520 tgttggtctt aaactgcaat agtagtttac cttgtattga agaaataaag accattttta 2580 tattaaaaaa tacttttgtc tgtcttcatt ttgacttgtc tgatatcctt gcagtgccca 2640 ttatgtcagt tctgtcagat attcagacat caaaacttaa cgtgagctca gtggagttac 2700 agctgcggtt ttgatgctgt tattatttct gaaactagaa atgatgttgt cttcatctgc 2760 tcatcaaaca cttcatgcag agtgtaaggc tagtgagaaa tgcatacatt tattgatact 2820 tttttaaagt caacttttta tcagattttt ttttcatttg gaaatatatt gttttctaga 2880 ctgcatagct tctgaatctg aaatgcagtc tgattggcat gaagaagcac agcactcttc 2940 atcttactta aacttcattt tggaatgaag gaagttaagc aagggcacag gtccatgaaa 3000 tagagacagt gcgctcagga gaaagtgaac ctggatttct ttggctagtg ttctaaatct 3060 gtagtgagga aagtaacacc cgattccttg aaagggctcc agctttaatg cttccaaatt 3120 gaaggtggca ggcaacttgg ccactggtta tttactgcat tatgtctcag tttcgcagct 3180 aacctggctt ctccactatt gagcatggac tatagcctgg cttcagaggc caggtgaagg 3240 ttgggatggg tggaaggagt gctgggctgt ggctgggggg actgtgggga ctccaagctg 3300 agcttggggt gggcagcaca gggaaaagtg tgggtaacta tttttaagta ctgtgttgca 3360 aacgtctcat ctgcaaatac gtagggtgtg tactctcgaa gattaacagt gtgggttcag 3420 taatatatgg atgaattcac agtggaagca ttcaagggta gatcatctaa cgacaccaga 3480 tcatcaagct atgattggaa gcggtatcag aagagcgagg aaggtaagca gtcttcatat 3540 gttttccctc cacgtaaagc agtctgggaa agtagcaccc cttgagcaga gacaaggaaa 3600 taattcagga gcatgtgcta ggagaacttt cttgctgaat tctacttgca agagctttga 3660 tgcctggctt ctggtgcctt ctgcagcacc tgcaaggccc agagcctgtg gtgagctgga 3720 gggaaagatt ctgctcaagt ccaagcttca gcaggtcatt gtctttgctt cttcccccag 3780 cactgtgcag cagagtggaa ctgatgtcga agcctcctgt ccactacctg ttgctgcagg 3840 cagactgctc tcagaaaaag agagctaact ctatgccata gtctgaaggt aaaatgggtt 3900 ttaaaaaaga aaacacaaag gcaaaaccgg ctgccccatg agaagaaagc agtggtaaac 3960 atggtagaaa aggtgcagaa gcccccaggc agtgtgacag gcccctcctg ccacctagag 4020 gcgggaacaa gcttccctgc ctagggctct gcccgcgaag tgcgtgtttc tttggtgggt 4080 tttgtttggc gtttggtttt gagatttaga cacaagggaa gcctgaaagg aggtgttggg 4140 cactattttg gtttgtaaag cctgtacttc aaatatatat tttgtgaggg agtgtagcga 4200 attggccaat ttaaaataaa gttgcaagag attgaaggct gagtagttga gagggtaaca 4260 cgtttaatga gatcttctga aactactgct tctaaacact tgtttgagtg gtgagacctt 4320 ggataggtga gtgctcttgt tacatgtctg atgcacttgc ttgtcctttt ccatccacat 4380 ccatgcattc cacatccacg catttgtcac ttatcccata tctgtcatat ctgacatacc 4440 tgtctcttcg tcacttggtc agaagaaaca gatgtgataa tccccagccg ccccaagttt 4500 gagaagatgg cagttgcttc tttccctttt tcctgctaag taaggatttt ctcctggctt 4560 tgacacctca cgaaatagtc ttcctgcctt acattctggg cattatttca aatatctttg 4620 gagtgcgctg ctctcaagtt tgtgtcttcc tactcttaga gtgaatgctc ttagagtgaa 4680 agagaaggaa gagaagatgt tggccgcagt tctctgatga acacacctct gaataatggc 4740 caaaggtggg tgggtttctc tgaggaacgg gcagcgtttg cctctgaaag caaggagctc 4800 tgcggagttg cagttatttt gcaactgatg gtggaactgg tgcttaaagc agattcccta 4860 ggttccctgc tacttctttt ccttcttggc agtcagttta tttctgacag acaaacagcc 4920 acccccactg caggcttaga aagtatgtgg ctctgcctgg gtgtgttaca gctctgccct 4980 ggtgaaaggg gattaaaacg ggcaccattc atcccaaaca ggatcctcat tcatggatca 5040 agctgtaagg aacttgggct ccaacctcaa aacattaatt ggagtacgaa tgtaattaaa 5100 actgcattct cgcattccta agtcatttag tctggactct gcagcatgta ggtcggcagc 5160 tcccactttc tcaaagacca ctgatggagg agtagtaaaa atggagaccg attcagaaca 5220 accaacggag tgttgccgaa gaaactgatg gaaataatgc atgaattgtg tggtggacat 5280 tttttttaaa tacataaact acttcaaatg aggtcggaga aggtcagtgt tttattagca 5340 gccataaaac caggtgagcg agtaccattt ttctctacaa gaaaaacgat tctgagctct 5400 gcgtaagtat aagttctcca tagcggctga agctcccccc tggctgcctg ccatctcagc 5460 tggagtgcag tgccatttcc ttggggtttc tctcacagca gtaatgggac aatacttcac 5520 aaaaattctt tcttttcctg tcatgtggga tccctactgt gccctcctgg ttttacgtta 5580 ccccctgact gttccattca gcggtttgga aagagaaaaa gaatttggaa ataaaacatg 5640 tctacgttat cacctcctcc agcattttgg tttttaatta tgtcaataac tggcttagat 5700 ttggaaatga gagggggttg ggtgtattac cgaggaacaa aggaaggctt atataaactc 5760 aagtctttta tttagagaac tggcaagctg tcaaaaacaa aaaggcctta ccaccaaatt 5820 aagtgaatag ccgctatagc cagcagggcc agcacgaggg atggtgcact gctggcacta 5880 tgccacggcc tgcttgtgac tctgagagca actgctttgg aaatgacagc acttggtgca 5940 atttcctttg tttcagaatg cgtagagcgt gtgcttggcg acagtttttc tagttaggcc 6000 acttcttttt tccttctctc ctcattctcc taagcatgtc tccatgctgg taatcccagt 6060 caagtgaacg ttcaaacaat gaatccatca ctgtaggatt ctcgtggtga tcaaatcttt 6120 gtgtgaggtc tataaaatat ggaagcttat ttatttttcg ttcttccata tcagtcttct 6180 ctatgacaat tcacatccac cacagcaaat taaaggtgaa ggaggctggt gggatgaaga 6240 gggtcttcta gctttacgtt cttccttgca aggccacagg aaaatgctga gagctgtaga 6300 atacagcctg gggtaagaag ttcagtctcc tgctgggaca gctaaccgca tcttataacc 6360 ccttctgaga ctcatcttag gaccaaatag ggtctatctg gggtttttgt tcctgctgtt 6420 cctcctggaa ggctatctca ctatttcact gctcccacgg ttacaaacca aagatacagc 6480 ctgaattttt tctaggccac attacataaa tttgacctgg taccaatatt gttctctata 6540 tagttatttc cttccccact gtgtttaacc ccttaaggca ttcagaacaa ctagaatcat 6600 agaatggttt ggattggaag gggccttaaa catcatccat ttccaaccct ctgccatggg 6660 ctgcttgcca cccactggct caggctgccc agggccccat ccagcctggc cttgagcacc 6720 tccagggatg gggcacccac agcttctctg ggcagcctgt gccaacacct caccactctc 6780 tgggtaaaga attctctttt aacatctaat ctaaatctct tctcttttag tttaaagcca 6840 ttcctctttt tcccgttgct atctgtccaa gaaatgtgta ttggtctccc tcctgcttat 6900 aagcaggaag tactggaagg ctgcagtgag gtctccccac agccttctct tctccaggct 6960 gaacaagccc agctccttca gcctgtcttc gtaggagatc atcttagtgg ccctcctctg 7020 gacccattcc aacagttcca cggctttctt gtggagcccc aggtctggat gcagtacttc 7080 agatggggcc ttacaaaggc agagcagatg gggacaatcg cttacccctc cctgctggct 7140 gcccctgttt tgatgcagcc cagggtactg ttggcctttc aggctcccag accccttgct 7200 gatttgtgtc aagcttttca tccaccagaa cccacgcttc ctggttaata cttctgccct 7260 cacttctgta agcttgtttc aggagacttc cattctttag gacagactgt gttacaccta 7320 cctgccctat tcttgcatat atacatttca gttcatgttt cctgtaacag gacagaatat 7380 gtattcctct aacaaaaata catgcagaat tcctagtgcc atctcagtag ggttttcatg 7440 gcagtattag cacatagtca atttgctgca agtaccttcc aagctgcggc ctcccataaa 7500 tcctgtattt gggatcagtt accttttggg gtaagctttt gtatctgcag agaccctggg 7560 ggttctgatg tgcttcagct ctgctctgtt ctgactgcac cattttctag atcacccagt 7620 tgttcctgta caacttcctt gtcctccatc ctttcccagc ttgtatcttt gacaaataca 7680 ggcctatttt tgtgtttgct tcagcagcca tttaattctt cagtgtcatc ttgttctgtt 7740 gatgccactg gaacaggatt ttcagcagtc ttgcaaagaa catctagctg aaaactttct 7800 gccattcaat attcttacca gttcttcttg tttgaggtga gccataaatt actagaactt 7860 cgtcactgac aagtttatgc attttattac ttctattatg tacttacttt gacataacac 7920 agacacgcac atattttgct gggatttcca cagtgtctct gtgtccttca catggtttta 7980 ctgtcatact tccgttataa ccttggcaat ctgcccagct gcccatcaca agaaaagaga 8040 ttcctttttt attacttctc ttcagccaat aaacaaaatg tgagaagccc aaacaagaac 8100 ttgtggggca ggctgccatc aagggagaga cagctgaagg gttgtgtagc tcaatagaat 8160 taagaaataa taaagctgtg tcagacagtt ttgcctgatt tatacaggca cgccccaagc 8220 cagagaggct gtctgccaag gccaccttgc agtccttggt ttgtaagata agtcataggt 8280 aacttttctg gtgaattgcg tggagaatca tgatggcagt tcttgctgtt tactatggta 8340 agatgctaaa ataggagaca gcaaagtaac acttgctgct gtaggtgctc tgctatccag 8400 acagcgatgg cactcgcaca ccaagatgag ggatgctccc agctgacgga tgctggggca 8460 gtaacagtgg gtcccatgct gcctgctcat tagcatcacc tcagccctca ccagcccatc 8520 agaaggatca tcccaagctg aggaaagttg ctcatcttct tcacatcatc aaacctttgg 8580 cctgactgat gcctcccgga tgcttaaatg tggtcactga catctttatt tttctatgat 8640 ttcaagtcag aacctccgga tcaggaggga acacatagtg ggaatgtacc ctcagctcca 8700 aggccagatc ttccttcaat gatcatgcat gctacttagg aaggtgtgtg tgtgtgaatg 8760 tagaattgcc tttgttattt tttcttcctg ctgtcaggaa cattttgaat accagagaaa 8820 aagaaaagtg ctcttcttgg catgggagga gttgtcacac ttgcaaaata aaggatgcag 8880 tcccaaatgt tcataatctc agggtctgaa ggaggatcag aaactgtgta tacaatttca 8940 ggcttctctg aatgcagctt ttgaaagctg ttcctggccg aggcagtact agtcagaacc 9000 ctcggaaaca ggaacaaatg tcttcaaggt gcagcaggag gaaacacctt gcccatcatg 9060 aaagtgaata accactgccg ctgaaggaat ccagctcctg tttgagcagg tgctgcacac 9120 tcccacactg aaacaacagt tcatttttat aggacttcca ggaaggatct tcttcttaag 9180 cttcttaatt atggtacatc tccagttggc agatgactat gactactgac aggagaatga 9240 ggaactagct gggaatattt ctgtttgacc accatggagt cacccatttc tttactggta 9300 tttggaaata ataattctga attgcaaagc aggagttagc gaagatcttc atttcttcca 9360 tgttggtgac agcacagttc tggctatgaa agtctgctta caaggaagag gataaaaatc 9420 atagggataa taaatctaag tttgaagaca atgaggtttt agctgcattt gacatgaaga 9480 aattgagacc tctactggat agctatggta tttacgtgtc tttttgctta gttacttatt 9540 gaccccagct gaggtcaagt atgaactcag gtctctcggg ctactggcat ggattgatta 9600 catacaactg taattttagc agtgatttag ggtttatgag tacttttgca gtaaatcata 9660 gggttagtaa tgttaatctc agggaaaaaa aaaaaaagcc aaccctgaca gacatcccag 9720 ctcaggtgga aatcaaggat cacagctcag tgcggtccca gagaacacag ggactcttct 9780 cttaggacct ttatgtacag ggcctcaaga taactgatgt tagtcagaag actttccatt 9840 ctggccacag ttcagctgag gcaatcctgg aattttctct ccgctgcaca gttccagtca 9900 tcccagtttg tacagttctg gcactttttg ggtcaggccg tgatccaagg agcagaagtt 9960 ccagctatgg tcagggagtg cctgaccgtc ccaactcact gcactcaaac aaaggcgaaa 10020 ccacaagagt ggcttttgtt gaaattgcag tgtggcccag aggggctgca ccagtactgg 10080 attgaccacg aggcaacatt aatcctcagc aagtgcaatt tgcagccatt aaattgaact 10140 aactgatact acaatgcaat cagtatcaac aagtggtttg gcttggaaga tggagtctag 10200 gggctctaca ggagtagcta ctctctaatg gagttgcatt ttgaagcagg acactgtgaa 10260 aagctggcct cctaaagagg ctgctaaaca ttagggtcaa ttttccagtg cactttctga 10320 agtgtctgca gttccccatg caaagctgcc caaacatagc acttccaatt gaatacaatt 10380 atatgcaggc gtactgcttc ttgccagcac tgtccttctc aaatgaactc aacaaacaat 10440 ttcaaagtct agtagaaagt aacaagcttt gaatgtcatt aaaaagtata tctgctttca 10500 gtagttcagc ttatttatgc ccactagaaa catcttgtac aagctgaaca ctggggctcc 10560 agattagtgg taaaacctac tttatacaat catagaatca tagaatggcc tgggttggaa 10620 gggaccccaa ggatcatgaa gatccaacac ccccgccaca ggcagggcca ccaacctcca 10680 gatctggtac tagaccaggc agcccagggc tccatccaac ctggccatga acacctccag 10740 ggatggagca tccacaacct ctctgggcag cctgtgccag cacctcacca ccctctctgt 10800 gaagaacttt tccctgacat ccaatctaag ccttccctcc ttgaggttag atccactccc 10860 ccttgtgcta tcactgtcta ctcttgtaaa aagttgattc tcctcctttt tggaaggttg 10920 caatgaggtc tccttgcagc cttcttctct tctgcaggat gaacaagccc agctccctca 10980 gcctgtcttt ataggagagg tgctccagcc ctctgatcat ctttgtggcc ctcctctgga 11040 cccgctccaa gagctccaca tctttcctgt actgggggcc ccaggcctga atgcagtact 11100 ccagatgggg cctcaaaaga gcagagtaaa gagggacaat caccttcctc accctgctgg 11160 ccagccctct tctgatggag ccctggatac aactggcttt ctgagctgca acttctcctt 11220 atcagttcca ctattaaaac aggaacaata caacaggtgc tgatggccag tgcagagttt 11280 ttcacacttc ttcatttcgg tagatcttag atgaggaacg ttgaagttgt gcttctgcgt 11340 gtgcttcttc ctcctcaaat actcctgcct gatacctcac cccacctgcc actgaatggc 11400 tccatggccc cctgcagcca gggccctgat gaacccggca ctgcttcaga tgctgtttaa 11460 tagcacagta tgaccaagtt gcacctatga atacacaaac aatgtgttgc atccttcagc 11520 acttgagaag aagagccaaa tttgcattgt caggaaatgg tttagtaatt ctgccaatta 11580 aaacttgttt atctaccatg gctgttttta tggctgttag tagtggtaca ctgatgatga 11640 acaatggcta tgcagtaaaa tcaagactgt agatattgca acagactata aaattcctct 11700 gtggcttagc caatgtggta cttcccacat tgtataagaa atttggcaag tttagagcaa 11760 tgtttgaagt gttgggaaat ttctgtatac tcaagagggc gtttttgaca actgtagaac 11820 agaggaatca aaagggggtg ggaggaagtt aaaagaagag gcaggtgcaa gagagcttgc 11880 agtcccgctg tgtgtacgac actggcaaca tgaggtcttt gctaatcttg gtgctttgct 11940 tcctgcccct ggctgcctta gggtgcgatc tgcctcagac ccacagcctg ggcagcagga 12000 ggaccctgat gctgctggct cagatgagga gaatcagcct gtttagctgc ctgaaggata 12060 ggcacgattt tggctttcct caagaggagt ttggcaacca gtttcagaag gctgagacca 12120 tccctgtgct gcacgagatg atccagcaga tctttaacct gtttagcacc aaggatagca 12180 gcgctgcttg ggatgagacc ctgctggata agttttacac cgagctgtac cagcagctga 12240 acgatctgga ggcttgcgtg atccagggcg tgggcgtgac cgagacccct ctgatgaagg 12300 aggatagcat cctggctgtg aggaagtact ttcagaggat caccctgtac ctgaaggaga 12360 agaagtacag cccctgcgct tgggaagtcg tgagggctga gatcatgagg agctttagcc 12420 tgagcaccaa cctgcaagag agcttgaggt ctaaggagta aaaagtctag agtcggggcg 12480 gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 12540 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 12600 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 12660 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa 12720 tcgataagga tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg 12780 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc 12840 gtaggacagg tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 12900 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 12960 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 13020 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 13080 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 13140 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 13200 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 13260 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 13320 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 13380 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 13440 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 13500 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 13560 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 13620 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 13680 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 13740 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 13800 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 13860 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 13920 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 13980 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 14040 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 14100 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 14160 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 14220 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 14280 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 14340 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 14400 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 14460 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 14520 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 14580 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 14640 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 14700 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 14760 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag 14820 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 14880 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 14940 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 15000 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 15060 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 15120 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 15180 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 15240 gtttacaatt tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 15300 ggcctcttcg ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg 15360 ggaggtactt ggagcggccg ctctagaact agtggatccc ccggccgcaa taaaatatct 15420 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct 15480 ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt 15540 gcaggtgcca gaacatttct ctatcgatag gtaccgagct cttacgcgtg ctagccctcg 15600 agcaggatct atacattgaa tcaatattgg caattagcca tattagtcat tggttatata 15660 gcataaatca atattggcta ttggccattg catacgttgt atctatatca taatatgtac 15720 atttatattg gctcatgtcc aatatgaccg ccatgttgac attgattatt gactagttat 15780 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca 15840 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 15900 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 15960 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg 16020 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 16080 ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg 16140 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 16200 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 16260 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 16320 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca 16380 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctcccctc gaagctcgac 16440 tctaggggct cgagatctgc gatctaagta agcttgcatg cctgcaggtc ggccgccacg 16500 accggtgccg ccaccatccc ctgacccacg cccctgaccc ctcacaagga gacgaccttc 16560 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt 16620 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga 16680 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga 16740 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga 16800 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg 16860 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga 16920 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg 16980 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct 17040 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc 17100 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg 17160 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atggctccga 17220 ccgaagccga cccgggcggc cccgccgacc ccgcacccgc ccccgaggcc caccgactct 17280 agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat gagtttggac 17340 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg 17400 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 17460 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca 17520 aatgtggtaa aatcgataag gatcaattcg gcttcaggta ccgtcgacga tgtaggtcac 17580 ggtctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc 17640 cacctcaccc atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc 17700 gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt 17760 gagcacggga cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt 17820 ctcgacggtc acggcgggca tgtcgacagc cgaattgatc cgtcgaccga tgcccttgag 17880 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact 17940 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt 18000 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 18060 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatg 18116 8 17402 DNA Plasmid pOMIFN-Ins-CMV-pur-attB 8 ggccgccacc gcggtggagc tccaattcgc cctatagtga gtcgtattac aattcactgg 60 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 120 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 180 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg 240 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 300 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 360 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 420 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 480 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 540 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 600 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 660 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 720 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 780 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 840 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 900 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 960 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 1020 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 1080 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1140 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1200 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1260 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 1320 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1380 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 1440 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 1500 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 1560 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 1620 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 1680 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 1740 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 1800 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 1860 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 1920 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 1980 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2040 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2100 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2160 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2220 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2280 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2340 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2400 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 2460 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 2520 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 2580 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 2640 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 2700 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 2760 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 2820 ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc tgggtaccgg 2880 gccccccctc gactagaggg acagcccccc cccaaagccc ccagggatgt aattacgtcc 2940 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 3000 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 3060 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg 3120 gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg 3180 ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac 3240 cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg 3300 gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact 3360 gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc 3420 tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca 3480 gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga 3540 catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa 3600 ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg gagaggagca 3660 caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg 3720 gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt cagtaggact 3780 gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa tcatgaaggc 3840 tggaaaagcc ctccaagatc cccaagacca accccaaccc acccaccgtg cccactggcc 3900 atgtccctca gtgccacatc cccacagttc ttcatcacct ccagggacgg tgaccccccc 3960 acctccgtgg gcagctgtgc cactgcagca ccgctctttg gagaaggtaa atcttgctaa 4020 atccagcccg accctcccct ggcacaacgt aaggccatta tctctcatcc aactccagga 4080 cggagtcagt gaggatgggg ctctagtcga ggtcgacggt atcgataagc ttgattaggc 4140 agagcaatag gactctcaac ctcgtgagta tggcagcatg ttaactctgc actggagtcc 4200 agcgtgggaa acaatctgcc ttgcacatga gtcttcgtgg gccaatattc cccaacggtt 4260 ttccttcagc ttgtcttgtc tcctaagctc tcaaaacacc tttttggtga ataaactcac 4320 ttggcaacgt ttatctgtct taccttagtg tcacgtttca tccctattcc cctttctcct 4380 cctccgtgtg gtacacagtg gtgcacactg gttcttctgt tgatgttctg ctctgacagc 4440 caatgtgggt aaagttcttc ctgccacgtg tctgtgttgt tttcacttca aaaagggccc 4500 tgggctcccc ttggagctct caggcatttc cttaatcatc acagtcacgc tggcaggatt 4560 agtccctcct aaaccttaga atgacctgaa cgtgtgctcc ctctttgtag tcagtgcagg 4620 gagacgtttg cctcaagatc agggtccatc tcacccacag ggccattccc aagatgaggt 4680 ggatggttta ctctcacaaa aagttttctt atgtttggct agaaaggaga actcactgcc 4740 tacctgtgaa ttcccctagt cctggttctg ctgccactgc tgcctgtgca gcctgtccca 4800 tggagggggc agcaactgct gtcacaaagg tgatcccacc ctgtctccac tgaaatgacc 4860 tcagtgccac gtgttgtata gggtataaag tacgggaggg ggatgcccgg ctcccttcag 4920 ggttgcagag cagaagtgtc tgtgtataga gtgtgtctta atctattaat gtaacagaac 4980 aacttcagtc ctagtgtttt gtgggctgga attgcccatg tggtagggac aggcctgcta 5040 aatcactgca atcgcctatg ttctgaaggt atttgggaaa gaaagggatt tgggggattg 5100 cctgtgattg gctttaattg aatggcaaat cacaggaaag cagttctgct caacagttgg 5160 ttgtttcagc caattcttgc agccaaagag ccgggtgccc agcgatataa tagttgtcac 5220 ttgtgtctgt atggatgaca gggaggtagg gtgacctgag gaccaccctc cagcttctgc 5280 tagcgtaggt acagtcacca cctccagctc cacacgagtc ccatcgtggt ttaccaaaga 5340 aacacaatta tttggaccag tttggaaagt cacccgctga attgtgaggc tagattaata 5400 gagctgaaga gcaaatgttc ccaacttgga gatactagtt ggtattagta tcagaggaac 5460 agggccatag cacctccatg ctattagatt ccggctggca tgtacttttc aagatgattt 5520 gtaactaaca atggcttatt gtgcttgtct taagtctgtg tcctaatgta aatgttcctt 5580 tggtttatat aaccttcttg ccatttgctc ttcaggtgtt cttgcagaac actggctgct 5640 ttaatctagt ttaactgttg cttgattatt cttagggata agatctgaat aaactttttg 5700 tggctttggc agactttagc ttgggcttag ctcccacatt agcttttgct gccttttctg 5760 tgaagctatc aagatcctac tcaatgacat tagctgggtg caggtgtacc aaatcctgct 5820 ctgtggaaca cattgtctga tgataccgaa ggcaaacgtg aactcaaaga ggcacagagt 5880 taagaagaag tctgtgcaat tcagaggaaa agccaaagtg gccattagac acactttcca 5940 tgcagcattt gccagtaggt ttcatataaa actacaaaat ggaataaacc actacaaatg 6000 ggaaaagcct gatactagaa tttaaatatt cacccaggct caaggggtgt ttcatggagt 6060 aatatcactc tataaaagta gggcagccaa ttattcacag acaaagcttt tttttttctg 6120 tgctgcagtg ctgtttttcg gctgatccag ggttacttat tgtgggtctg agagctgaat 6180 gatttctcct tgtgtcatgt tggtgaagga gatatggcca gggggagatg agcatgttca 6240 agaggaaacg ttgcattttg gtggcttggg agaaaggtag aacgatatca ggtccatagt 6300 gtcactaaga gatctgaagg atggttttac agaacagttg acttggctgg gtgcaggctt 6360 ggctgtaaat ggatggaagg atggacagat gggtggacag agatttctgt gcaggagatc 6420 atctcctgag ctcggtgctt gacagactgc agatccatcc cataaccttc tccagcatga 6480 gagcgcgggg agctttggta ctgttcagtc tgctgcttgt tgcttcctgg gtgcacagtg 6540 gtgattttct tactcacaca gggcaaaaac ctgagcagct tcaaagtgaa caggttgctc 6600 tcataggcca ttcagttgtc aagatgaggt ttttggtttc ttgttttgta aggtgggaag 6660 aagcactgaa ggatcagttg cgagggcagg ggtttagcac tgttcagaga agtcttattt 6720 taactcctct catgaacaaa aagagatgca ggtgcagatt ctggcaagca tgcagtgaag 6780 gagaaagccc tgaatttctg atatatgtgc aatgttgggc acctaacatt ccccgctgaa 6840 gcacagcagc tccagctcca tgcagtactc acagctggtg cagccctcgg ctccagggtc 6900 tgagcagtgc tgggactcac gaggttccat gtctttcaca ctgataatgg tccaatttct 6960 ggaatgggtg cccatccttg gaggtcccca aggccaggct ggctgcgtct ccgagcagcc 7020 cgatctggtg gtgagtagcc agcccatggc aggagttaga gcctgatggt ctttaaggtc 7080 ccttccaacc taagccatcc tacgattcta ggaatcatga cttgtgagtg tgtattgcag 7140 aggcaatatt ttaaagttat aaatgttttc tccccttcct tgtttgtcaa agttatcttg 7200 atcgccttat caatgctttt ggagtctcca gtcatttttc ttacamcaaa aagaggagga 7260 agaatgaaga gaatcattta atttcttgat tgaatagtag gattcagaaa gctgtacgta 7320 atgccgtctc tttgtatcga gctgtaaggt ttctcatcat ttatcagcgt ggtacatatc 7380 agcacttttc catctgatgt ggaaaaaaaa atccttatca tctacagtct ctgtacctaa 7440 acatcgctca gactctttac caaaaaagct ataggtttta aaactacatc tgctgataat 7500 ttgccttgtt ttagctcttc ttccatatgc tgcgtttgtg agaggtgcgt ggatgggcct 7560 aaactctcag ctgctgagct tgatgggtgc ttaagaatga agcactcact gctgaaactg 7620 ttttcatttc acaggaatgt tttagtggca ttgtttttat aactacatat tcctcagata 7680 aatgaaatcc agaaataatt atgcaaactc actgcatccg ttgcacaggt ctttatctgc 7740 tagcaaagga aataatttgg ggatggcaaa aacattcctt cagacatcta tatttaaagg 7800 aatataatcc tggtacccac ccacttcatc cctcattatg ttcacactca gagatactca 7860 ttctcttgtt gttatcattt gatagcgttt tctttggttc tttgccacgc tctgggctat 7920 ggctgcacgc tctgcactga tcagcaagta gatgcgaggg aagcagcagt gagaggggct 7980 gccctcagct ggcacccagc cgctcagcct aggaggggac cttgcctttc caccagctga 8040 ggtgcagccc tacaagctta cacgtgctgc gagcaggtga gcaaagggag tcttcatggt 8100 gtgtttcttg ctgcccggaa gcaaaacttt actttcattc attccccttg aagaatgagg 8160 aatgtttgga aacggactgc tttacgttca atttctctct tccctttaag gctcagccag 8220 gggccattgc tgaggacggc atcggggccc cctggaccaa atctgtggca cagatggttt 8280 cacttacatc agtggatgtg ggatctgcgc ctgtaatgtg tccttctgaa ggaaggaacg 8340 tgccttccaa gtgccagccc cacagccccc agcccctccc tgtgctgctc caattcatct 8400 cctcttcctc cttctccctt tgctgtttgt gctcgggtag aaatcatgaa gatttagaag 8460 agaaaacaaa ataactggag tggaaaccca ggtgatgcag ttcattcagc tgtcataggt 8520 ttgtcgttgc tataggtctg tatcagagat gctarcacca ctttgctgtc ggtgcttaac 8580 tcgggtgaac tctccttcac tcgcatcatt tgcgggcctt atttacatcc ccagcatcca 8640 tcaccctctg ggaaaatggg cgcactggat ctctaatgga agactttccc tctttcagag 8700 cctgtgggat gtgcagtgac aagaaacgtg gaggggctga gcagcagcac tgcccccagg 8760 gagcaggagc ggatgccatc ggtggcagca tcccaaatga tgtcagcgga tgctgagcag 8820 gcagcggacg aacggacaga agcgatgcgt acaccttctg ttgacatggt atttggcagc 8880 gatttaacac tcgcttccta gtcctgctat tctccacagg ctgcattcaa atgaacgaag 8940 ggaagggagg caaaaagatg caaaatccga gacaagcagc agaaatattt cttcgctacg 9000 gaagcgtgcg caaacaacct tctccaacag caccagaaga gcacagcgta acctttttca 9060 agaccagaaa aggaaattca caaagcctct gtggatacca gcgcgttcag ctctcctgat 9120 agcagatttc ttgtcaggtt gcgaatgggg tatggtgcca ggaggtgcag ggaccatatg 9180 atcatataca gcacagcagt cattgtgcat gtattaatat atattgagta gcagtgttac 9240 tttgccaaag caatagttca gagatgagtc ctgctgcata cctctatctt aaaactaact 9300 tataaatagt aaaaccttct cagttcagcc acgtgctcct ctctgtcagc accaatggtg 9360 cttcgcctgc acccagctgc aaggaatcag cccgtgatct cattaacact cagctctgca 9420 ggataaatta gattgttcca ctctcttttg ttgttaatta cgacggaaca attgttcagt 9480 gctgatggtc ctaattgtca gctacagaaa acgtctccat gcagttcctt ctgcgccagc 9540 aaactgtcca ggctatagca ccgtgatgca tgctacctct cactccatcc ttcttctctt 9600 tcccaccagg gagagctgtg tgttttcact ctcagccact ctgaacaata ccaaactgct 9660 acgcactgcc tccctcggaa agagaatccc cttgttgctt ttttatttac aggatccttc 9720 ttaaaaagca gaccatcatt cactgcaaac ccagagcttc atgcctctcc ttccacaacc 9780 gaaaacagcc ggcttcattt gtctttttta aatgctgttt tccaggtgaa ttttggccag 9840 cgtgttggct gagatccagg agcacgtgtc agctttctgc tctcattgct cctgttctgc 9900 attgcctctt tctggggttt ccaagagggg gggagacttt gcgcggggat gagataatgc 9960 cccttttctt agggtggctg ctgggcagca gagtggctct gggtcactgt ggcaccaatg 10020 ggaggcacca gtgggggtgt gttttgtgca ggggggaagc attcacagaa tggggctgat 10080 cctgaagctt gcagtccaag gctttgtctg tgtacccagt gaaatccttc ctctgttaca 10140 taaagcccag ataggactca gaaatgtagt cattccagcc cccctcttcc tcagatctgg 10200 agcagcactt gtttgcagcc agtcctcccc aaaatgcaca gacctcgccg agtggaggga 10260 gatgtaaaca gcgaaggtta attacctcct tgtcaaaaac actttgtggt ccatagatgt 10320 ttctgtcaat cttacaaaac agaaccgaga ggcagcgagc actgaagagc gtgttcccat 10380 gctgagttaa tgagacttgg cagctcgctg tgcagagatg atccctgtgc ttcatgggag 10440 gctgtaacct gtctccccat cgccttcaca ccgcagtgct gtcctggaca cctcaccctc 10500 cataagctgt aggatgcagc tgcccaggga tcaagagact tttcctaagg ctcttaggac 10560 tcatctttgc cgctcagtag cgtgcagcaa ttactcatcc caactatact gaatgggttt 10620 ctgccagctc tgcttgtttg tcaataagca tttcttcatt ttgcctctaa gtttctctca 10680 gcagcaccgc tctgggtgac ctgagtggcc acctggaacc cgaggggcac agccaccacc 10740 tccctgttgc tgctgctcca gggactcatg tgctgctgga tggggggaag catgaagttc 10800 ctcacccaga cacctgggtt gcaatggctg cagcgtgctc ttcttggtat gcagattgtt 10860 tccagccatt acttgtagaa atgtgctgtg gaagcccttt gtatctcttt ctgtggccct 10920 tcagcaaaag ctgtgggaaa gctctgaggc tgctttcttg ggtcgtggag gaattgtatg 10980 ttccttcttt aacaaaaatt atccttagga gagagcactg tgcaagcatt gtgcacataa 11040 aacaattcag gttgaaaggg ctctctggag gtttccagcc tgactactgc tcgaagcaag 11100 gccaggttca aagatggctc aggatgctgt gtgccttcct gattatctgt gccaccaatg 11160 gaggagattc acagccactc tgcttcccgt gccactcatg gagaggaata ttcccttata 11220 ttcagataga atgttatcct ttagctcagc cttccctata accccatgag ggagctgcag 11280 atccccatac tctccccttc tctggggtga aggccgtgtc ccccagcccc ccttcccacc 11340 ctgtgcccta agcagcccgc tggcctctgc tggatgtgtg cctatatgtc aatgcctgtc 11400 cttgcagtcc agcctgggac atttaattca tcaccagggt aatgtggaac tgtgtcatct 11460 tcccctgcag ggtacaaagt tctgcacggg gtcctttcgg ttcaggaaaa ccttcactgg 11520 tgctacctga atcaagctct atttaataag ttcataagca catggatgtg ttttcctaga 11580 gatacgtttt aatggtatca gtgattttta tttgctttgt tgcttacttc aaacagtgcc 11640 tttgggcagg aggtgaggga cgggtctgcc gttggctctg cagtgatttc tccaggcgtg 11700 tggctcaggt cagatagtgg tcactctgtg gccagaagaa ggacaaagat ggaaattgca 11760 gattgagtca cgttaagcag gcatcttgga gtgatttgag gcagtttcat gaaagagcta 11820 cgaccactta ttgttgtttt ccccttttac aacagaagtt ttcatcaaaa taacgtggca 11880 aagcccagga atgtttggga aaagtgtagt taaatgtttt gtaattcatt tgtcggagtg 11940 ctaccagcta agaaaaaagt cctacctttg gtatggtagt cctgcagaga atacaacatc 12000 aatattagtt tggaaaaaaa caccaccacc accagaaact gtaatggaaa atgtaaacca 12060 agaaattcct tgggtaagag agaaaggatg tcgtatactg gccaagtcct gcccagctgt 12120 cagcctgctg accctctgca gttcaggacc atgaaacgtg gcactgtaag acgtgtcccc 12180 tgcctttgct tgcccacaga tctctgccct tgtgctgact cctgcacaca agagcatttc 12240 cctgtagcca aacagcgatt agccataagc tgcacctgac tttgaggatt aagagtttgc 12300 aattaagtgg attgcagcag gagatcagtg gcagggttgc agatgaaatc cttttctagg 12360 ggtagctaag ggctgagcaa cctgtcctac agcacaagcc aaaccagcca agggttttcc 12420 tgtgctgttc acagaggcag ggccagctgg agctggagga ggttgtgctg ggacccttct 12480 ccctgtgctg agaatggagt gatttctggg tgctgttcct gtggcttgca ctgagcagct 12540 caagggagat cggtgctcct catgcagtgc caaaactcgt gtttgatgca gaaagatgga 12600 tgtgcacctc cctcctgcta atgcagccgt gagcttatga aggcaatgag ccctcagtgc 12660 agcaggagct gtagtgcact cctgtaggtg ctagggaaaa tctctggttc ccagggatgc 12720 attcataagg gcaatatatc ttgaggctgc gccaaatctt tctgaaatat tcatgcgtgt 12780 tcccttaatt tatagaaaca aacacagcag aataattatt ccaatgcctc ccctcgaagg 12840 aaacccatat ttccatgtag aaatgtaacc tatatacaca cagccatgct gcatccttca 12900 gaacgtgcca gtgctcatct cccatggcaa aatactacag gtattctcac tatgttggac 12960 ctgtgaaagg aaccatggta agaaacttcg gttaaaggta tggctgcaaa actactcata 13020 ccaaaacagc agagctccag acctcctctt aggaaagagc cacttggaga gggatggtgt 13080 gaaggctgga ggtgagagac agagcctgtc ccagttttcc tgtctctatt ttctgaaacg 13140 tttgcaggag gaaaggacaa ctgtactttc aggcatagct ggtgccctca cgtaaataag 13200 ttccccgaac ttctgtgtca tttgttctta agatgctttg gcagaacact ttgagtcaat 13260 tcgcttaact gtgactaggt ctgtaaataa gtgctccctg ctgataaggt tcaagtgaca 13320 tttttagtgg tatttgacag catttacctt gctttcaagt cttctaccaa gctcttctat 13380 acttaagcag tgaaaccgcc aagaaaccct tccttttatc aagctagtgc taaataccat 13440 taacttcata ggttagatac ggtgctgcca gcttcacctg gcagtggttg gtcagttctg 13500 ctggtgacaa agcctccctg gcctgtgctt ttacctagag gtgaatatcc aagaatgcag 13560 aactgcatgg aaagcagagc tgcaggcacg atggtgctga gccttagctg cttcctgctg 13620 ggagatgtgg atgcagagac gaatgaagga cctgtccctt actcccctca gcattctgtg 13680 ctatttaggg ttctaccaga gtccttaaga ggtttttttt ttttttggtc caaaagtctg 13740 tttgtttggt tttgaccact gagagcatgt gacacttgtc tcaagctatt aaccaagtgt 13800 ccagccaaaa tcaattgcct gggagacgca gaccattacc tggaggtcag gacctcaata 13860 aatattacca gcctcattgt gccgctgaca gattcagctg gctgctccgt gttccagtcc 13920 aacagttcgg acgccacgtt tgtatatatt tgcaggcagc ctcgggggga ccatctcagg 13980 agcagagcac cggcagccgc ctgcagagcc gggcagtacc tcaccatggc tttgaccttt 14040 gccttactgg tggctctcct ggtgctgagc tgcaagagca gctgctctgt gggctgcgat 14100 ctgcctcaga cccacagcct gggcagcagg aggaccctga tgctgctggc tcagatgagg 14160 agaatcagcc tgtttagctg cctgaaggat aggcacgatt ttggctttcc tcaagaggag 14220 tttggcaacc agtttcagaa ggctgagacc atccctgtgc tgcacgagat gatccagcag 14280 atctttaacc tgtttagcac caaggatagc agcgctgctt gggatgagac cctgctggat 14340 aagttttaca ccgagctgta ccagcagctg aacgatctgg aggcttgcgt gatccagggc 14400 gtgggcgtga ccgagacccc tctgatgaag gaggatagca tcctggctgt gaggaagtac 14460 tttcagagga tcaccctgta cctgaaggag aagaagtaca gcccctgcgc ttgggaagtc 14520 gtgagggctg agatcatgag gagctttagc ctgagcacca acctgcaaga gagcttgagg 14580 tctaaggagt aaaaagtcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 14640 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 14700 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 14760 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 14820 agcaagtaaa acctctacaa atgtggtaaa atcgataccg tcgacctcga ctagagcggc 14880 cactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg 14940 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtaccg agctcttacg 15000 cgtgctagcc ctcgagcagg atctatacat tgaatcaata ttggcaatta gccatattag 15060 tcattggtta tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat 15120 atcataatat gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat 15180 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 15240 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 15300 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 15360 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 15420 atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 15480 cccagtacat gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg 15540 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 15600 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 15660 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 15720 ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct 15780 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc 15840 cctcgaagct cgactctagg ggctcgagat ctgcgatcta agtaagcttg catgcctgca 15900 ggtcggccgc cacgaccggt gccgccacca tcccctgacc cacgcccctg acccctcaca 15960 aggagacgac cttccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 16020 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 16080 accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 16140 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 16200 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 16260 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg 16320 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 16380 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg 16440 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 16500 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 16560 aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga 16620 ccccatggct ccgaccgaag ccgacccggg cggccccgcc gaccccgcac ccgcccccga 16680 ggcccaccga ctctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat 16740 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 16800 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 16860 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 16920 gtaaaacctc tacaaatgtg gtaaaatcga taaggatcaa ttcggcttca ggtaccgtcg 16980 acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc cttgggctcc 17040 ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc gaggtggcgg 17100 tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa accgctgggc 17160 gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg cgggtgcgga tacgcggggc 17220 agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga cagccgaatt gatccgtcga 17280 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact 17340 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca 17400 gc 17402 9 5172 DNA Plasmid pRSV-Int 9 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 60 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 120 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 180 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 240 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 300 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 360 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 420 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 480 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 540 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 600 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 660 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 720 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 780 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 840 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 900 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 960 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1020 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1080 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1140 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1200 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1260 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1320 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1380 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1440 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1500 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1560 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1620 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1680 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 1740 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 1800 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 1860 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 1920 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 1980 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa 2040 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 2100 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 2160 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 2220 cgcgtgctag gggtctagga tcgattctag gaattctcta gccgcggtct agggatcccg 2280 gcgcgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 2340 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 2400 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 2460 gcttcgcgat gtacgggcca gatatacgcg tatctgaggg gactagggtg tgtttaggcg 2520 aaaagcgggg cttcggttgt acgcggttag gagtcccctc aggatatagt agtttcgctt 2580 ttgcataggg agggggaaat gtagtcttat gcaatacact tgtagtcttg caacatggta 2640 acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg 2700 gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacaggtct gacatggatt 2760 ggacgaacca ctgaattccg cattgcagag ataattgtat ttaagtgcct agctcgatac 2820 aataaacgcc atttgaccat tcaccacatt ggtgtgcacc tccaagcttg catgcctgca 2880 ggtaccggtc cggaattccc gggtcgacga gctcactagt cgtagggtcg ccgacatgac 2940 acaaggggtt gtgaccgggg tggacacgta cgcgggtgct tacgaccgtc agtcgcgcga 3000 gcgcgagaat tcgagcgcag caagcccagc gacacagcgt agcgccaacg aagacaaggc 3060 ggccgacctt cagcgcgaag tcgagcgcga cgggggccgg ttcaggttcg tcgggcattt 3120 cagcgaagcg ccgggcacgt cggcgttcgg gacggcggag cgcccggagt tcgaacgcat 3180 cctgaacgaa tgccgcgccg ggcggctcaa catgatcatt gtctatgacg tgtcgcgctt 3240 ctcgcgcctg aaggtcatgg acgcgattcc gattgtctcg gaattgctcg ccctgggcgt 3300 gacgattgtt tccactcagg aaggcgtctt ccggcaggga aacgtcatgg acctgattca 3360 cctgattatg cggctcgacg cgtcgcacaa agaatcttcg ctgaagtcgg cgaagattct 3420 cgacacgaag aaccttcagc gcgaattggg cgggtacgtc ggcgggaagg cgccttacgg 3480 cttcgagctt gtttcggaga cgaaggagat cacgcgcaac ggccgaatgg tcaatgtcgt 3540 catcaacaag cttgcgcact cgaccactcc ccttaccgga cccttcgagt tcgagcccga 3600 cgtaatccgg tggtggtggc gtgagatcaa gacgcacaaa caccttccct tcaagccggg 3660 cagtcaagcc gccattcacc cgggcagcat cacggggctt tgtaagcgca tggacgctga 3720 cgccgtgccg acccggggcg agacgattgg gaagaagacc gcttcaagcg cctgggaccc 3780 ggcaaccgtt atgcgaatcc ttcgggaccc gcgtattgcg ggcttcgccg ctgaggtgat 3840 ctacaagaag aagccggacg gcacgccgac cacgaagatt gagggttacc gcattcagcg 3900 cgacccgatc acgctccggc cggtcgagct tgattgcgga ccgatcatcg agcccgctga 3960 gtggtatgag cttcaggcgt ggttggacgg cagggggcgc ggcaaggggc tttcccgggg 4020 gcaagccatt ctgtccgcca tggacaagct gtactgcgag tgtggcgccg tcatgacttc 4080 gaagcgcggg gaagaatcga tcaaggactc ttaccgctgc cgtcgccgga aggtggtcga 4140 cccgtccgca cctgggcagc acgaaggcac gtgcaacgtc agcatggcgg cactcgacaa 4200 gttcgttgcg gaacgcatct tcaacaagat caggcacgcc gaaggcgacg aagagacgtt 4260 ggcgcttctg tgggaagccg cccgacgctt cggcaagctc actgaggcgc ctgagaagag 4320 cggcgaacgg gcgaaccttg ttgcggagcg cgccgacgcc ctgaacgccc ttgaagagct 4380 gtacgaagac cgcgcggcag gcgcgtacga cggacccgtt ggcaggaagc acttccggaa 4440 gcaacaggca gcgctgacgc tccggcagca aggggcggaa gagcggcttg ccgaacttga 4500 agccgccgaa gccccgaagc ttccccttga ccaatggttc cccgaagacg ccgacgctga 4560 cccgaccggc cctaagtcgt ggtgggggcg cgcgtcagta gacgacaagc gcgtgttcgt 4620 cgggctcttc gtagacaaga tcgttgtcac gaagtcgact acgggcaggg ggcagggaac 4680 gcccatcgag aagcgcgctt cgatcacgtg ggcgaagccg ccgaccgacg acgacgaaga 4740 cgacgcccag gacggcacgg aagacgtagc ggcgtagcga gacacccgga tccctcgagg 4800 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct 4860 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 4920 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 4980 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 5040 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagg 5100 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc 5160 actgggggcg cg 5172 10 6233 DNA Plasmid pCR-XL-TOPO-CMV-pur-attB 10 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 240 ttaggtgacg cgttagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca 300 ctagtaacgg ccgccagtgt gctggaattc gcccttggcc gcaataaaat atctttattt 360 tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat 420 caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt 480 gccagaacat ttctctatcg ataggtaccg agctcttacg cgtgctagcc ctcgagcagg 540 atctatacat tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa 600 atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat 660 attggctcat gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag 720 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 780 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 840 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 900 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct 960 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg 1020 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 1080 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 1140 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 1200 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 1260 tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 1320 tttgacctcc atagaagaca ccgggaccga tccagcctcc cctcgaagct cgactctagg 1380 ggctcgagat ctgcgatcta agtaagcttg catgcctgca ggtcggccgc cacgaccggt 1440 gccgccacca tcccctgacc cacgcccctg acccctcaca aggagacgac cttccatgac 1500 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac 1560 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca 1620 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg 1680 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt 1740 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg 1800 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc 1860 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc 1920 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac 1980 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt 2040 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc 2100 gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct ccgaccgaag 2160 ccgacccggg cggccccgcc gaccccgcac ccgcccccga ggcccaccga ctctagagtc 2220 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca 2280 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 2340 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 2400 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg 2460 gtaaaatcga taaggatcaa ttcggcttca ggtaccgtcg acgatgtagg tcacggtctc 2520 gaagccgcgg tgcgggtgcc agggcgtgcc cttgggctcc ccgggcgcgt actccacctc 2580 acccatctgg tccatcatga tgaacgggtc gaggtggcgg tagttgatcc cggcgaacgc 2640 gcggcgcacc gggaagccct cgccctcgaa accgctgggc gcggtggtca cggtgagcac 2700 gggacgtgcg acggcgtcgg cgggtgcgga tacgcggggc agcgtcagcg ggttctcgac 2760 ggtcacggcg ggcatgtcga cagccgaatt gatccgtcga ccgatgccct tgagagcctt 2820 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 2880 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc 2940 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3000 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg aagggcgaat 3060 tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg cccaattcgc 3120 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 3180 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 3240 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta tacgtacggc 3300 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga 3360 gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt gcacgtctgc 3420 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc 3480 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg 3540 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat 3600 aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc acgtagaaag 3660 ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa 3720 gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc 3780 tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg 3840 gtaaggttgg gaagccctgc aaagtaaact ggatggcttt ctcgccgcca aggatctgat 3900 ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc atgattgaac 3960 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 4020 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 4080 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg 4140 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 4200 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 4260 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 4320 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 4380 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 4440 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc 4500 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 4560 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 4620 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 4680 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 4740 tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg 4800 gtatttcaca ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4860 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4920 gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt 4980 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc 5040 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat 5100 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg 5160 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc 5220 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga 5280 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact 5340 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 5400 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 5460 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5520 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5580 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 5640 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5700 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5760 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5820 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 5880 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 5940 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6000 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6060 caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca tgttctttcc 6120 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 6180 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag 6233 11 234 DNA artificial attP containing polynucleotide 11 gactagtact gacggacaca ccgaagcccc ggcggcaacc ctcagcggat gccccggggc 60 ttcacgtttt cccaggtcag aagcggtttt cgggagtagt gccccaactg gggtaacctt 120 tgagttctct cagttggggg cgtagggtcg ccgacatgac acaaggggtt gtgaccgggg 180 tggacacgta cgcgggtgct tacgaccgtc agtcgcgcga gcgcgactag taca 234 12 26 DNA artificial Primer attB-for 12 taccgtcgac gatgtaggtc acggtc 26

Claims

What is claimed is:

1. A method for genetically transforming an avian cell, comprising:

delivering to an avian cell having a first recombination site a nucleic acid molecule comprising a second recombination site;

delivering a source of integrase activity to the avian cell; and

maintaining the avian cell under conditions suitable for the integrase to mediate recombination between the first and the second recombination sites, thereby integrating the nucleic acid molecule into the nuclear genome of the avian cell.

2. The method of claim 1, wherein the avian cell is selected from the group consisting of a stage I cell, a stage X blastodermal cell, a primordial germline cell and an oviduct cell.

3. The method of claim 1, wherein the integrase is a serine recombinase

4. The method of claim 1, wherein the integrase is the bacteriophage phiC31 integrase.

5. The method of claim 1, wherein the source of integrase activity is delivered to the avian cell as a polypeptide.

6. The method of claim 1, wherein the source of integrase activity is expressed from a polynucleotide molecule delivered to the avian cell.

7. The method of claim 6, wherein the polynucleotide molecule is an mRNA.

8. The method of claim 6, wherein the polynucleotide molecule is an expression vector.

9. The method of claim 8, wherein the expression vector comprises a promoter operably linked to a region encoding the integrase, wherein the promoter is selected from a CMV promoter and an RSV promoter.

10. The method of claim 9, wherein the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 1 and 9.

11. The method of claim 1, wherein the nucleic acid molecule comprises an isolated gene expression control region operably linked to a region encoding a polypeptide selected to be expressed by an avian cell.

12. The method of claim 1, wherein the nucleic acid molecule is an expression vector.

13. The method of claim 12, wherein the expression vector is selected from the group consisting of a plasmid, an artificial chromosome, an isolated eukaryotic chromosome, an adenovirus vector and a lentivirus vector.

14. The method of claim 11, wherein the gene expression control region is isolated from an avian gene selected from the group consisting of an ovalbumin gene, a lysozyme gene and an ovomucoid gene.

15. The method of claim 11, wherein the nucleic acid molecule further comprises an IRES operably linked to a region encoding a second polypeptide selected to be expressed by an avian cell.

16. The method of claim 11, wherein the region encoding the polypeptide is codon-optimized for expression of the polypeptide by the avian cell.

17. The method of claim 15 wherein the region encoding the second polypeptide is codon-optimized for expression of the polypeptide by the avian cell.

18. The method of claim 1, wherein the first recombination site is a attP site having at least 25% identity to the sequence according to SEQ ID NO: 11.

19. The method of claim 1, wherein the first recombination site is a attP site having at least 75% identity to the sequence according to SEQ ID NO: 11.

20. The method of claim 1, wherein the first recombination site is a attP site according to SEQ ID NO: 11.

21. The method of claim 1, wherein the first recombination site is a pseudo-attP site.

22. The method of claim 1, wherein the nucleic acid molecule and the source of integrase activity are sequentially delivered to the isolated avian cell.

23. The method of claim 1, wherein the nucleic acid molecule and the source of integrase activity are concurrently delivered to the isolated avian cell.

24. The method of claim 1, wherein the avian cell is a chicken cell.

25. The method of claim 1, wherein the avian cell is a quail cell.

26. The method of claim 1, wherein the nucleic acid molecule and the source of integrase activity are delivered to the avian cell by a method or combination of methods selected from injection, electroporation, DNA-PEI, virus-PEI, microcell fusion, artificial viral coats, polylysine conjugation and lipofection.

27. The method of claim 1, wherein the avian cell is an oviduct cell in the oviduct of an avian and further comprising the steps of: before delivering the nucleic acid molecule and source of integrase activity, surgically exposing the luminal surface of the oviduct of an avian;

delivering the nucleic acid molecule and the source of integrase activity to the oviduct cell by depositing the nucleic acid molecule and the source of integrase activity on the luminal surface;

applying an electroporation pulse across the wall of the oviduct, wherein the electroporation pulse is directed to deliver the nucleic acid molecule and the source of integrase activity to recipient oviduct cells, whereby the integrase mediates integration of the nucleic acid molecule into the genome of a recipient cell;

surgically closing the oviduct; and

maintaining the avian under conditions suitable for the expression by the recipient oviduct cells of a polypeptide encoded by the nucleic acid molecule and deposition of said polypeptide into the white of a laid hard shell egg.

28. A genetically transformed avian cell, and progeny thereof, generated by a of the methods according to claims 1.

29. A genetically transformed avian, and progeny thereof, generated by the method according to claim 27.

30. A method for generating a genetically transformed avian, comprising:

genetically transforming an avian cell by a method according to claim 1;

delivering the genetically transformed avian cell to an early stage avian embryo, thereby generating a genetically transformed embryo; and

maintaining the avian embryo under conditions suitable for the embryo to develop and hatch as a transgenic chick.

31. The method of claim 26, wherein the genetically transformed embryo is delivered to a recipient female avian and laid as a hard shell egg.

32. A method of producing a polypeptide comprising expressing the polypeptide in a transgenic avian cell, said cell comprising a heterologous nucleic acid molecule that expresses the polypeptide, wherein the heterologous nucleic acid molecule is integrated into the nuclear genome and flanked by halves of an integration site recognized by an integrase.

33. A polypeptide of interest expressed by a genetically transformed avian cell, and progeny thereof, the cell comprising a heterologous nucleic acid molecule integrated into the nuclear genome thereof by an integrase, and wherein the nucleic acid molecule comprises a region encoding the polypeptide of interest and an operably linked promoter region.

34. The polypeptide according to claim 33, wherein the polypeptide is an immunoglobulin chain.

35. The polypeptide according to claim 33, wherein the polypeptide is a cytokine.

36. The polypeptide according to claim 33, wherein the polypeptide is deposited in the white of an avian egg.

37. The polypeptide according to claim 33, wherein the polypeptide is expressed by a transgenic bird comprising the genetically transformed avian cell.

38. The polypeptide according to claim 37, wherein the genetically transformed avian cell, and the progeny thereof, are generated according to the methods of claim 1.

39. The polypeptide according to claim 37, wherein the transgenic bird is generated according to the method of claim 30.

40. An avian egg white comprising a heterologous polypeptide produced according to the method according to the claim 32.

41. An avian egg comprising a heterologous polypeptide produced according to the method according to the claim 32.

42. A method for generating a genetically modified avian cell, and progeny thereof, using a tagged chromosome, said method comprising the steps of:

providing an isolated modified chromosome comprising a lac operator region and a first recombination site;

delivering the modified chromosome to a avian cell, thereby generating a trisomic avian cell;

delivering to the avian cell a source of a tagged polypeptide comprising a fluorescent domain and a lac repressor domain;

delivering a source of integrase activity to the avian cell;

delivering a polynucleotide comprising a second recombination site and a region encoding a polypeptide to the avian cell;

maintaining the avian cell under conditions suitable for the integrase to mediate recombination between the first and second recombination sites, thereby integrating the polynucleotide into the modified chromosome and generating a genetically modified avian cell;

expressing the tag polypeptide by the avian cell;

allowing the tag polypeptide to bind to the modified chromosome so as to label the modified chromosome; and

isolating the modified chromosome by selecting modified chromosomes having a tag polypeptide bound thereto.

43. The method according to claim 42, further comprising the step of delivering the modified chromosome to a second avian cell to generate a trisomic avian cell.

44. A modified avian chromosome comprising a lac operator region and a recombination site.

45. The avian chromosome according to claim 44, wherein the lac operator region is a concatamer of lac operators.

46. The avian chromosome according to claim 44, wherein the recombination site is selected from an att B or an att P site.

47. The avian chromosome according to claim 44, further comprising a region encoding a tag polypeptide and an operably linked promoter.

48. The avian chromosome according to claim 47, wherein the tag polypeptide comprises a fluorescent domain and a lac repressor domain.

49. The avian chromosome according to claim 44, wherein the florescent domain is GFP.

50. The avian chromosome according to claim 45, further comprising a heterologous polynucleotide integrated into the first recombination site.

51. The avian chromosome according to claim 50, wherein the heterologous polynucleotide comprises a region encoding a polypeptide operably linked to a promoter.

52. The avian chromosome according to claim 50, wherein the promoter is an avian promoter selected from the gene expression control region of an onomucoid gene, a lysozyne gene and an ovalbumin gene.